Pluripotent stem cell that can be isolated from body tissue

ABSTRACT

Objects of the present invention are to provide a method for directly obtaining pluripotent stern cells which do not have tumorigenic property from body tissue and the thus obtained pluripotent stem cells. The present invention relates to SSEA-3 (+) pluripotent stern cells that can be isolated from body tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/189,215, filed Jun. 22, 2016, which is a Divisional of U.S.application Ser. No. 13/435,703, filed Mar. 30, 2012, now U.S. Pat. No.9,399,758, which is a Continuation-In-Part of U.S. application Ser. No.12/836,264, filed Jul. 14, 2010, now U.S. Pat. No. 9,550,975, whichclaims the benefits of priority to U.S. Provisional Application Nos.61/290,159, filed Dec. 24, 2009, and 61/213,788, filed Jul. 15, 2009,and to Japanese Patent Application Nos. JP 2011-076643, filed Mar. 30,2011, and JP 2011-076635, filed Mar. 30, 2011. The entire contents ofall of the above applications are incorporated herein by reference.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 25, 2016, isnamed sequence.txt and is 3 KB.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to body tissue-derived Pluripotent stemcells.

Background Art

Planarians and newts can regenerate their entire bodies even after theirbodies have been cut. Such high regenerative capacity of planarians andnewts depends on the presence of pluripotent stem cells existing in themesenchymal tissues. However, in the case of higher organisms such ashumans, the tissue regenerative capacity is far lower than that of thoseanimals. Cells of inner cell mass in a mammalian blastocyst isrecognized as pluripotent stern cells that is capable of differentiatinginto cells of ectodermal, mesodermal, and endodermal cell lineages.However, such broad spectrum of differentiation ability becomes limitedas development proceeds, followed by cell differentiation forspecialization resulting in each type of tissue, and thus, unlikeplanarians and newts, pluripotent stem cell are not considered to beresiding in the living body of higher mammals.

Bone marrow stromal cell (MSC) fractions are known to having the abilityto differentiate into bone, cartilage, adipocytes, neuronal cells andskeletal muscles by induction, and the like have been reported as cellshaving differentiation potency obtained from an adult (see Non-PatentDocuments 1 and 2). However, bone marrow stromal cells (MSCs) arecomprised of various cell kinds, and thus cells responsible for thedifferentiation of MSC fraction are not clearly understood. Furthermore,induction of MSCs requires stimulation with a specific compound, genetransfer, or the like for differentiation of MSCs into specific cells.Specifically, there is a need to construct a system for inducingdifferentiation.

Furthermore, there have been reported that when MSCs are infused intoliving body, a small part of MSC fractions are known to home intodamaged tissues and differentiate into tissue specific cells andcontribute to repair. Such tissue repair was observed in tissues andorgans of endodermal-, ectodermal- and mesodermal origins. However, thecells responsible for the repair remained an enigma.

iPS cells (induced pluripotent stem cells) (see Patent document 1,Patent document 2, Non-patent document 3, and the like) have beenreported as adult-derived pluripotent stem cells. However, establishmentof iPS cells requires an artificial operation using a specificsubstance, such as introduction of a specific gene into mesenchymal cellfraction (for example, a dermal fibroblast fraction) or introduction ofa specific compound into somatic cells. While iPS can generate cell ofall three germ layers, their tumorigenicity pose limitation for clinicalappliation.

Patent document 1 JP Patent No. 4183742

Patent document 2 JP Patent Publication (Kokai) No. 2008-307007 A

Non-patent document 1 M. DEZAWA et al., The Journal of ClinicalInvestigation, 113, 12, pp. 1701-1710, (2004)

Non-patent document 2 M. DEZAWA et al., SCIENCE, 2005 July 8, 309, pp.314-317, (2005)

Non-patent document 3 Okita K. et al. SCIENCE, 2008 Nov 7, 322 (5903),pp.949-953

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for directlyobtaining pluripotent stem cells from body tissue and pluripotent stemcells obtained by the method. As described in “Background Art”, thereare pluripotent stem cells existing in mesenchymal tissues in some loweranimals. The present inventors newly found that the novel type ofpluripotent stem cells can be isolated from body tissues of a livinganimals without artificial manipulation such as introduction ofexogenous genes. The expressed antigens of the novel pluripotent stemcells differ from those of the conventionally reported pluripotent stemcells. The pluripotent stem cells can self-renew and can differentiateinto endodermal, ectodermal, and mesodermal cells whereas they do nothave tumorigenicity. Accordingly, the present invention enables thatpluripotent stem cells with higher safety can be isolated from bodytissues of a human.

The present inventors have discovered that in the research process ofculturing bone marrow stromal cell (MSC: bone marrow stromal cell)fractions in ordinary adherent culture, characteristic cell clusters arespontaneously formed at very low frequency from untreated (naive) humanand rodent MSC cells. The appearance of initial cell clusters closelyresembles that of ES cell-derived embryoid body. However, unlike EScells, the cell clusters do not undergo infinite growth. They stopgrowth when they reach a size within a certain period and then they formheterogeneous populations containing various cells such as hair cellsand pigment cells. Together with hairs and pigment cells, cells positivefor ectodermal, endodermal, and mesodermal markers were shown to becontained in a single cell cluster by immunocytochemistry. The presentinventors considered from the results the possible presence of cellsequivalent to pluripotent cells in untreated (naive) human MSC cellfractions that are generally maintained and cultured. The presentinventors then further intensively studied the matter.

The present inventors have discovered that SSEA-3 is expressed as asurface antigen of the above pluripotent stern cells and that the abovepluripotent stern cells can also be isolated from body tissue usingSSEA-3 expression as a marker. The present inventors further found thatthe pluripotent stem cells can also be isolated from body tissue usingboth of SSEA-3 and CD105 expressions as markers. SSEA-3 is a pluripotentstem cell marker and CD105 is a mesenchymal marker. The pluripotent stemcells of the present invention can be isolated by using SSEA-3 only as amarker. However, when the pluripotent stem cells are isolated fromtissues which contain elements other than mesenchymal lineage, combineuse of SSEA-3 and CD105 is effective.

When isolated SSEA-3(+) were cultured in suspension either in singlecell suspension culture or in methylcellulose (MC)-containing medium(called “MC culture”), single SSEA-3(+) cells start to proliferate, formcharacteristic cell cluster (from 25 μm to up to a maximum diameter of150 μm) resembling embryoid body of human ES cells. The cluster expresspluripotency markers such as Nanog, Oct3/4 and Sox2, was positive foralkaline phosphatase reaction. Importantly, the cluster formed from asingle SSEA-3(+) cell generated ectodermal-, endodermal- andmesodermal-lineage cells when transferred onto gelatin culture to inducespontaneous differentiation, demonstrating that SSEA-3(+) cell has theability to generate cells representative of all three germ layers.Furthermore, SSEA-3(+) cell showed the ability to self-renew. A“pluripotent stem cell” is defined as that having the ability to giverise to cell types of all three embryonic germ layers, namelyendodermal, mesodermal, and ectodermal cells from a single cells, andthat having the ability to self-renew, the invented cell is pluripotentstem cell.

It is known that when a body is exposed to stress or injured, tissuestem cells in a dormant state are activated, contributing to tissueregeneration. The present inventors have provided stimulation stress tomesenchymal cells such as bone marrow stromal cell (MSC) fractions anddermal fibroblast fractions while culturing them according to variousmethods (e.g., serum free culture, culture using Hank's Balanced SaltSolution (HBSS), low oxygen culture, a total of 3 hours of intermittentshort-time trypsin culture, and 8 or 16 hours of long-time trypsinincubation), collected surviving cells, and then performed suspensionculture in MC culture. As a result, formation of embryoid body-like cellclusters (up to a maximum diameter of 150 μm) was confirmed as describedabove. In particular, embryoid body-like cell cluster formation wasconfirmed at the highest frequency in human dermal fibroblast fractionsand human MSC fractions subjected to long-term trypsin incubation. Theproperty of formed cluster by stress stimulation was identical to thoseof SSEA-3(+) cells, thus the stress stimulation is found to be effectivemethod to enrich the pluripotent stem cells.

The present inventors have also discovered that the above pluripotentstem cells are novel type of pluripotent stem cells that differ fromconventionally reported pluripotent stem cells such as ES cells and iPScells. Specifically, the present inventors have discovered that thepluripotent stem cells can be directly obtained from body tissue withoutany induction operation. The present inventors have also discovered thatthe pluripotent stem cells show low or no telomerase activity and do notshow tumorigenic proliferative activity.

The present inventors have further discovered that cells in the obtainedembryoid body-like cell clusters had properties which were not possessedby the pluripotent stem cells which have been previously reported. Thepresent inventors have further examined proteins expressed by cells inthe obtained cell clusters, and thus they have discovered that the cellsexert expression patterns differing from those exerted by conventionallyreported pluripotent stem cells such as ES cells and iPS cells.

Thus, the present inventors have completed the present invention. Thepresent inventors have designated the pluripotent stem cells Muse cells(Multilineage-differentiating Stress Enduring cells).

The present invention is as follows.

-   [1] A SSEA-3-positive pluripotent stem cell, which can be isolated    from body tissue.

The pluripotent stem cells can be isolated from a culture product ofbody tissue such as cultured fibroblasts and myeloid stem cells and canalso be isolated in the form of single cells.

-   [2] The pluripotent stem cell according to [1], which is positive    for mesenchymal marker CD105.-   [3] The pluripotent stem cell according to [1] or [2], which is    negative for CD117 (c-Kit) and negative for CD146.-   [4] The pluripotent stem cell according to [1] or [2], which is    negative for CD117, negative for CD146, negative for NG2, negative    for CD34, negative for vWF, and negative for CD271.-   [5] The pluripotent stem cell according to [1] or [2], which is    negative for CD34, negative for CD117, negative for CD146, negative    for CD271, negative for NG2, negative for vWF, negative for Sox10,    negative for Snai1, negative for Slug, negative for Tyrp1, and    negative for Dct.-   [6] The pluripotent stem cell according to any one of [1] to [5],    which has low or no telomerase activity.-   [7-1] The pluripotent stem cell according to any one of [1] to [6],    which is capable of differentiating into the three germ layers.

The pluripotent stem cells of the present invention are capable ofdifferentiating into the three germ layers through in vitro adherentculture. Specifically, the pluripotent stem cells can differentiate intocells representative of the three germ layers, skin, liver, nerve,muscle, bone, fat, and the like through in vitro induction culture.Also, the pluripotent stem cells are capable of differentiating intocells characteristic of the three germ layers when transplanted in vivo;the pluripotent stem cells are capable of surviving and differentiatinginto organs (e.g., skin, spinal cord, liver, and muscle) whentransplanted to the damaged organs via intravenous injection into aliving body.

-   [7-2] The pluripotent stem cells can act as tissue repairing cells.    They integrate as functional cells into damaged tissue and    differentiate into ectodermal-, endodermal-, and mesodermal-lineage    cells according to the site of integration, and contribute to tissue    reconstruction.-   [8] The pluripotent stem cell according to any one of [1] to [7],    which does not undergo tumorigenic proliferation.

The pluripotent stern cells of the present invention have a propertysuch that they grow at a growth rate of about 1.3 days/cell division bysuspension culture but stop the growth within about 10 days and alsohave a property such that when transplanted into the testis, they do notbecome tumorigenic for at least a half year.

-   [9] The pluripotent stem cell according to any one of [1] to [8],    which has self-renewal capability.

The pluripotent stem cells of the present invention can be grown throughadherent culture. The pluripotent stem cells of the present inventioncan also be grown through repetition of suspension culture and adherentculture.

-   [10] The pluripotent stem cell according to any one of [1] to [9],    which is resistant to stress.-   [11] The pluripotent stem cell according to any one of [1] to [10],    which has high phagocytic ability.-   [12] The pluripotent stern cell according to any one of [1] to [11],    which is positive for at least one of the 22 following odorant    receptors:-   olfactory receptor, family 8, subfamily G, member 2 (OR8G2);-   olfactory receptor, family 7, subfamily G, member 3 (OR7G3);-   olfactory receptor, family 4, subfamily D, member 5 (OR4D5);-   olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);-   olfactory receptor, family 10, subfamily H, member 4 (ORIOH4);-   olfactory receptor, family 10, subfamily T, member 2 (OR10T2);-   olfactory receptor, family 2, subfamily M, member 2 (OR2M2);-   olfactory receptor, family 2, subfamily T, member 5 (OR2T5);-   olfactory receptor, family 7, subfamily D, member 4 (OR7D4);-   olfactory receptor, family 1, subfamily L, member 3 (OR1L3);-   olfactory receptor, family 4, subfamily N, member 4 (OR4N4);-   olfactory receptor, family 2, subfamily A, member 7 (OR2A7);-   guanine nucleotide binding protein (G protein), alpha activating    activity polypeptide,-   olfactory type (GNAL);-   olfactory receptor, family 6, subfamily A, member 2 (OR6A2);-   olfactory receptor, family 2, subfamily B, member 6 (OR2B6);-   olfactory receptor, family 2, subfamily C, member 1 (OR2C1);-   olfactory receptor, family 52, subfamily A, member 1 (OR52A1);-   olfactory receptor, family 10, subfamily H, member 3 (OR1OH3);-   olfactory receptor, family 10, subfamily H, member 2 (OR1OH2);-   olfactory receptor, family 51, subfamily E, member 2 (OR51E2);-   olfactory receptor, family 5, subfamily P, member 2 (OR5P2); and-   olfactory receptor, family 10, subfamily P, member 1 (OR10P1).-   [13] The pluripotent stem cell according to any one of [1] to [12],    which is positive for at least one of the 5 following chemokine    receptors:-   chemokine (C-C motif) receptor 5 (CCR5);-   chemokine (C-X-C motif) receptor 4 (CXCR4);-   chemokine (C-C motif) receptor 1 (CCR1);-   Duffy blood group, chemokine receptor (DARC); and-   chemokine (C-X-C motif) receptor 7 (CXCR7).-   [14] The pluripotent stem cell according to any one of [1] to [13],    which is derived from mesodermal tissue or mesenchymal tissue.-   [15] A cell cluster or a cell fraction, which contains the    pluripotent stem cell according to any one of [1] to [14].-   [16] A method for isolating a pluripotent stem cell or a pluripotent    cell fraction from body tissue, which uses at least one of the    following properties (i) to (vi) as an index:-   (i) being positive for SSEA-3;-   (ii) being positive for CD105;-   (iii) being negative for CD117 and negative for CD146;-   (iv) being negative for CD117, negative for CD146, negative for NG2,    negative for CD34, negative for vWF, and negative for CD271;-   (v) being negative for CD34, negative for CD117, negative for CD146,    negative for CD271, negative for NG2, negative for vWF, negative for    SoxlO, negative for Snai1, negative for Slug, negative for Tyrp1,    and negative for Dct; and-   (vi) having low or no telomerase activity.-   [17] A method for enrichment of a pluripotent stem cell or a    pluripotent cell fraction, which comprises exposing body    tissue-derived cells to cellular stress and then collecting    surviving cells.-   [18] The method for enrichment of a pluripotent stem cell or a    pluripotent cell fraction according to [17], wherein cellular stress    is selected from among protease treatment, culture under low-oxygen    conditions, culture under low phosphate conditions, culture under    serum starvation conditions, culture in a sugar starvation state,    culture under exposure to radiation, culture under exposure to heat    shock, culture in the presence of a toxic substance, culture in the    presence of active oxygen, culture under mechanical stimulation, and    culture under pressure treatment.-   [19] The method for enrichment of a pluripotent stem cell or a    pluripotent cell fraction according to [18], wherein the cellular    stress is trypsin incubation.-   [20] A pluripotent stem cell, which is a cell derived or induced    from the pluripotent stem cell according to any one of [1] to [14].

Examples of the derived cell or the induced cell include cells inducedby gene transfer or addition of a compound. Another example thereof isan iPS cell from the stem cell of the present invention.

-   [21] A differentiated cell, which is a cell derived or induced from    the pluripotent stem cell according to any one of [1] to [14].-   [22] A pharmaceutical composition, which comprises the pluripotent    stem cell according to any one of [1] to [14] and [20].-   [23] A pharmaceutical composition, which comprises the    differentiated cell according to [21].-   [24] A pharmaceutical composition, which comprises the    differentiated cell according to [21].-   [25] The pluripotent stem cell according to [1], which is derived    from umbilical cord or fat tisse of a living body.-   [26] A cell therapy composition for allo-transplantation comprising    the pluripotent stem cell according to [1], in which the pulipotent    stem cell does not express HLA class II antigen.-   [27] A method for regenerate a tissue in a subject, which comprises    administering the the pluripotent stem cell according to [1], in    which the pulipotent stem cell does not express HLA class II    antigen.

The specification includes part or all of the contents as disclosed inthe specifications and/or drawings of U.S. Provisional Application No.61/213,788 and U.S. Provisional Application No. 61/290,159, which arepriority documents of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relationship among mesenchymal cell fractions, Musecells, and M-clusters (Muse cell-derived embryoid body-like cellclusters). As shown in FIG. 1, SSEA-3-postive cells are directlyseparated and then cultured by suspension culture without exposure tolong-term stress, so that M-clusters can be obtained. M-clustergenerates ectodermal, endodermal and mesodermal cells on gelatinculture.

FIG. 2 shows a method for causing the growth of Muse cells in largeamounts.

FIG. 3 shows factors, the ratio of the expression level thereof in aM-cluster to that in a naive cell fraction was high.

FIG. 4 shows factors, the ratio of the expression level thereof in aM-cluster to that in human ES cells was high.

FIG. 5 shows protocols for MACS sorting.

FIGS. 6A and 6B show photos (stained images) showing the removal of deadcells when human fibroblast (H-fibroblast) fractions were subjected to16-hr-long trypsin incubation (FIG. 6A), 3 minutes of vortexing at 1800rpm-2200 rpm/minute (FIG. 6B), and then trypan blue staining.

FIGS. 7A, 7B, 7C, 7D, 7E, 7F and 7G show photos of various cells. FIG.7A shows a single cell (Bar=10 μm) in a Muse-e nriched cell fraction,FIG. 7B shows a human ES cell-derived embryoid body cell cluster (Bar=25μm), FIG. 7C shows a M-cluster (Bar=25 μm) with a diameter of about 25μm, FIG. 7D shows a human ES-derived cell cluster (on day 4) stained byalkaline phosphatase staining (Bar=25 μm), and FIGS. 7E-7G showimmunologically stained images of Oct3/4(e), Sox2(f), and PAR4(g) inM-clusters.

FIGS. 8A, 8B, 8C, 8D, 8E and 8F show photos showing the characteristicsof cell clusters formed from general adherent culture of H-fibroblastsand human MSCs (H-MSCs). FIGS. 8A and 8B show cell clusters (Bar=100 μm)spontaneously formed by general adherent culture for naive human MSCs.FIGS. 8C and 8D show the state of an H-fibroblast-1 fraction on day 0(c) and day 7 (d) subjected to long-term trypsin incubation followed byMC culture (Bar=100 μm). An arrow head in FIG. 8D indicates a M-cluster.FIGS. 8E and 8F show M-clusters formed from an H-fibroblast-1 fractionon day 7 of MC culture (Bar=50 μm).

FIGS. 9A, 9B, 9C, 9D, 9E and 9F show photos showing the characteristicsof cell clusters from Muse-enriched cell fraction from H-fibroblasts andhuman MSCs (H-MSCs). FIGS. 9A-9F show the results of immunostaining;that is, show the localization of Nanog (FIGS. 9A and 9D), Oct3/4 (FIGS.9B), SSEA-3 (FIG. 9C), PAR4 (FIG. 9E), and Sox2 (FIG. 9F) in M-clusters(FIGS. 9A, C, and 9E) formed from H-fibroblasts and M-clusters (FIGS.9B, D, and 9F) formed from H-MSCs (Bar=50 μm).

FIGS. 10A, 10B and 10C show photos showing the characteristics of cellclusters formed from Muse-enriched cell fractions ofs H-fibroblasts andhuman MSCs. FIGS. 10A-C show the results of alkaline phosphatasestaining for human ES cells (FIG. 10A), M-clusters (FIG. 10B) fromH-fibroblasts, and naive H-fibroblast-1 in adherent culture (FIG. 10C)(Bar=50 μm).

FIGS. 11A, 11B and 11C show electron micrographs showing thecharacteristics of cell clusters from H-fibroblasts and human MSCs(H-MSCs). FIGS. 11A-C show electron microscopic images of human ES cellembryoid bodies (FIG. 11A, on day 3 of MC culture), H-fibroblast-1-derived M-clusters (FIGS. 11B and 11C, on day 5 of MC culture)(Bar=5μm).

FIG. 12 demonstrates the clonality and self-renewal of M-clusters.Specifically, FIG. 12 shows the outline of an experiment conducted fordetermination of the clonality and self-renewal of Muse cells.

FIG. 13 shows the growth rate of Muse cells in suspension culture.

FIG. 14 shows a normal karyotype of cells clonally expanded from asingle M-cluster (H-fibroblast-1-derived,1^(st) generation (cycle)).

FIGS. 15A, 15B and 15C show the differentiation of M-clusters. FIGS.15A-15C show immunologically stained images showing the localization ofα smooth muscle actin and neurofilaments (FIGS. 15A and 15B) andα-fetoprotein (FIG. 15C) in differentiated M-clusters fromH-fiboroblast-1 (Bar=500 μm in FIG. 15A; Bar=50 μm in FIGS. 15B and15C). Arrow heads in FIG. 15A indicate adhered M-cluster.

FIG. 16 shows the results of RT-PCR analysis for α-fetoprotein (α-FP)expression, GATA6 expression, MAP-2 expression, and Nkx2.5 expression incell populations prepared by culturing a naive cell fraction, and1^(st)-generation and 3^(rd)-generation M-clusters from H-fibroblasts ongelatin so as to induce spontaneous differentiation. As positivecontrols, human fetus liver was used for α-FP and whole human embryoswere used for GATA6, MAP-2, and Nkx2.5.

FIGS. 17 show the testis of immunodeficient mice to which aMuse-enriched cell fraction was administered. FIG. 17 shows a controlintact testis, a testis obtained via administration of mouse ES cells(mES cells) (week 8), a testis obtained via administration of MEF(feeder cells) (week 8), a testis obtained via administration of aM-cluster (M-clusters) (month 6), and a testis obtained viaadministration of a Muse-enriched cell fraction (Muse) (month 6).

FIG. 18 shows the results of quantitative PCR for factors involved inpluripotency and undifferentiated cell states of H-fibroblasts (Fibro-1and Fibro-2) and H-MSCs (MSC-1 and MSC-2) (No. 2). Each pattern given ina column in FIG. 18 indicates the result of comparing the geneexpression level in Muse-enriched cell fractions or M-clusters (day 7)with the same in naive cell fractions. A white pattern indicates thatthe ratio of the gene expression level in the Muse-enriched cellfractions or the M-clusters to the same in naive cell fractions isgreater than 1/3 (1:3) but is lower than 3 (3:1). A gray patternindicates that the ratio of the gene expression level in theMuse-enriched cell fractions or the M-clusters to the same in naive cellfractions is greater than 3 (3:1). A pattern of oblique lines indicatesthat the ratio of the gene expression level in the Muse-enriched cellfractions or the M-clusters to the same in naive cell fractions is lowerthan 1/3 (1:3).

FIG. 19 shows the telomerase activity of H-MSC-derived naive cellfractions (Naive), Muse-enriched cell fractions (Muse), and M-clusters(formed in suspension day 7). Heat-inactivated samples (Heat) were usedas negative controls.

FIG. 20 shows the results of DNA microarray analysis for H-fibroblast-and H-MSC -derived naive cell fractions (referred to as “naive”),Muse-enriched cell fractions (referred to as “Muse”), and M-clusters(referred to as “EB”).

FIGS. 21A and 21B show photos showing embryoid body-like cell clustersformed by MC culture of Muse cells directly collected as SSEA-3/CD105double positive cells from a mononuclear cell component of human bonemarrow aspirate. FIG. 21A shows M-clusters formed by performing MCculture (7 days) for mononuclear cell fractions isolated from human bonemarrow (Bar=100 μm). FIG. 21B shows an alkaline phosphatase-stainedimage of M-cluster (Bar=50 μm).

FIG. 22 shows the results of RT-PCR analysis for α-fetoprotein (α-FP),GATA6, MAP-2, and Nkx2.5 in cell fractions prepared by culturing ongelatin M-clusters formed from naive H-MSC-1 (naive 1), naive H-MSC-2(naive 2) (both fractions were negative controls), and human bonemarrow-derived mononuclear cell fractions (8 hr-hBM) subjected to 8hours of trypsin incubation or human bone marrow-derived mononuclearcell fractions (naive hBM) not subjected to trypsin incubation, so as toinduce spontaneous differentiation thereof.

FIG. 23 shows the results of FACS analysis for H-fibroblasts (naivecells) and H-MSCs (naive cells).

FIGS. 24A and 24B show photos showing: a stained image of SSEA-3 (+)cells (left in 24A) in naive cells; and a stained image of SSEA-3 (+)cells (right in 24B) that clonally expanded from single M-clusters fromSSEA-3 (+) cells collected by FACS sorting. Each bar in this figureindicates 100 μm.

FIG. 25 shows photos of stained images showing the localization ofNumb-like (green) that is a factor involved in asymmetric divisionduring cell division of Muse cells (H-fibroblasts). Each bar in thisfigure indicates 100 μm.

FIG. 26A and 26B show electron micrographs showing H-fibroblast-derivedSSEA-3 (−) cells (FIG. 26A) and SSEA-3 (+) cells (FIG. 26B). Each bar inthis figure indicates 5 μm.

FIGS. 27A, 27B and 27C show photos of stained images showing Oct3/4(green) (FIG. 27A), Sox2 (green) (FIG. 27B), and SSEA-3 (red) (FIG. 27C)in H-fibroblast-derived Muse cells.

FIGS. 28A, 28B and 28C show photos showing the differentiation ofGFP-labeled SSEA-3 (+) Muse cell fractions in damaged tissue of severelyimmunodeficient mice (Nog mice). FIGS. 28A and B show GFP (+) cells of aspinal cord damaged due to compression (4 weeks later), expressingneurofilaments (red) and human golgi complexes (white). FIG. 28B showsan enlarged image of a part enclosed by a square in FIG. 28A. FIG. 28Cshows GFP (+) target cells of a damaged liver (4 weeks later),expressing human albumin (red) and human golgi complexes (white).

FIG. 29 shows photos showing the expression of human albumin in theliver into which SSEA-3 (+) Muse cells were transplanted, as examined byRT-PCR.

FIG. 30 shows photos showing the differentiation of GFP-labeled SSEA-3(+) Muse cell fractions in damaged tissue of severely immunodeficientmice (Nog mice). Specifically, the photos show GFP (+) cells of muscle(3 weeks later) expressing human dystrophin (red) in the damaged miceskeletal muscle.

FIGS. 31A, 31B, 31C, 31D, 31E, 31F, 31G, 31H, 31I and 31J show photosshowing the differentiation of cells grown from M-clusters formed fromsingle Muse cells. FIGS. 31A-D show the results of neural induction.FIG. 31A shows the thus formed neurospheres. Furthermore, asimmunostaining data for neurospheres, FIG. 31B shows the expression ofnestin, FIG. 31C shows the expression of Musashi, and FIG. 31D shows theexpression of NuroD. FIG. 31E shows MAP-2 (+) cells obtained by furthercausing these spheres to differentiate into neural cells. FIGS. 31F-Gshow the results of osteocyte induction and specifically show theexpression of osteocalcin (F) and alkalinephosphatase (G). FIGS. 31H andI show the results of adipocyte induction. FIG. 31H shows cellscontaining lipid droplets and FIG. 31I shows the result of oil redstaining. FIGS. 31J shows the result of hepatocyte induction; that is,α-fetoprotein (+) cells.

FIG. 32 shows photos showing the expression of human albumin and humanα-fetoprotein in cells induced by hepatocyte induction from Muse cells,as examined by RT-PCR. h-fibroblast is negative control and h-fetusliver is positive control.

FIG. 33 shows photos showing the expression of Sox10, Snai1, Slug,Tyrp1, and Dct in SSEA-3 (+) Muse cells obtained from H-fibroblasts, asexamined by RT-PCR. “Embryo” is positive control.

FIGS. 34A, 34B, 34C, 34D, 34E and 34F show the expression of NG2, CD34,vWF, CD117, CD146, and CD271, as analyzed by FACS. In naive human dermalfibroblasts, NG2 that is a pericyte marker and CD34 and vWF that areendothelial progenitor cell markers were found to be negative. They werealso found to be negative in SSEA-3 (+) cells. Few naive human dermalfibroblasts were found to be positive for CD117 that is a melanoblastmarker, CD146 that is a pericyte marker, and CD271 that is a NCSC marker(0.2%, 0.2%, and 0.8%, respectively), but were not thought to be Musecells since they were SSEA-3 (−) cells.

FIGS. 35A and 35B show that a Muse cell phagocytized ferrite.

FIGS. 36A, 36B, 36C, 36D, 36E and 36F show photos showing the formationof iPS cells prepared from Muse cells. FIG. 36A shows a state of humaniPS cells induced from dermal fibroblast -derived Muse cells. FIGS.36B-F show the expression of pluripotent cell markers (“b” shows theexpression of Nonog, “c” shows the expression of Oct3/4, “d” shows theexpression of Sox2, “e” shows the expression of SSEA-3, and “f′ showsthe expression of Tra-1-60).

FIGS. 37A, 37B, 37C and 37D show photos showing the results ofimmunohistochemical analysis of Muse cell-derived iPS cells for Nonog(A), Oct3/4 (B), Sox2 (C), and Tra-1-81 (D).

FIG. 38 shows photos showing the expression of pluripotency markers incolonies ((−)−1 and (−)−2) from Muse-derived iPS cells (Mi-1 and Mi-2)and SSEA-3 (−) cell-derived non-iPS colonies, as examined by RT-PCR.

FIGS. 39A, 39B, 39C and 39D show photos showing the results of Tra-1-81immunostaining of colonies formed by SSEA-3 (+) and (−) cells on day 30after culturing on MEF feeder cells following introduction of Oct3/4,Sox2, Klf4, and c-Myc with retrovirus. Human ES cells were used ascontrols. Colonies (a1) from SSEA-3 (+) cells and human ES cells (a2)were positive for Tra-1-81, but all colonies from SSEA-3 (−) cells werenegative for the same.

FIG. 40 shows photos showing the expression of pluripotency markers(endogenous Oct3/4 (endo Oct), endogenous Sox2 (endo Sox2), Nanog,endogenous Klf4 (endo Klf4), Rexl, and UTF1) for SSEA-3 (+) and (−)cells at a stage after 30 days of culture on MEF as in FIG. 39. In theSSEA-3 (-) cell fraction, no Sox2 and Nanog signals were observed.

FIGS. 41A, 41B, 41C and 41D show photos showing iPS cell colony (Musecell-derived iPS cells) (FIGS. 41A and B) and non-iPS cell colonygenerated from SSEA-3 (−) cells (FIGS. 41C and D).

FIGS. 42A and 42B show photos showing in vitro differentiation of iPScells induced from dermal fibroblast-derived Muse cells. FIG. 42A showsthe expression of oc-fetoprotein (green) that is an endodermal markerand smooth muscle actin (red (blue indicates DNA)) that is a mesodermalmarker. FIG. 42B shows the expression of neurofilament (green) that isan ectodermal marker.

FIG. 43 shows the results of RT-PCR analysis for in vitrodifferentiation of iPS cells induced from dermal fibroblast-derived Musecells. FIG. 43 specifically shows the expression of markers for the 3germ layers.

FIGS. 44A, 44B, 44C, 44D, 44E and 44F show photos showing tissuestructures of teratomas formed from iPS cells induced from dermalfibroblast -derived Muse cells. FIG. 44 specifically showsdifferentiation of iPS cells into various types of tissue, as revealedby HE (Hematoxylin and eosin) staining. FIG. 44B shows cartilage, FIG.44C shows muscle, FIG. 44D shows neural epithelium, FIG. 44E showspigmented epithelium, and FIG. 44F shows columnar epithelium.

FIG. 45 shows the results of bisulfite (hydrogensulfite) sequencing inMuse cells for the Nanog gene and the Oct3/4 gene of naive state,M-clusters, and Muse-derived iPS cells. The numerical value in eachcolumn indicates the position of CpG downstream of the transcriptionstart site (TSS). An open circle indicates unmethylated cytosine and afilled circle indicates methylated cytosine.

FIG. 46 shows the results of quantitative PCR for factors involved inthe cell cycle of naive fibroblasts (Naive), M-clusters (Cluster), andiPS cells (iPS). Among columns denoted with “/Naive,” open columnsindicate that the ratio of Muse fractions or M-clusters to naive cellsis less than 2 (2:1) and higher than 1/2 (1:2). Also, filled columnsindicate that the same ratio is higher than 2 (2:1). Columns shaded withoblique lines indicate that the same ratio is lower than 1/2 (1:2).Among columns denoted with “/iPS,” Symbol “*” indicates that the geneexpression level in M-clusters is higher than that in iPS cells. Symbol“*” indicates that the gene expression level in iPS cells is higher thanthat in M-clusters.

FIG. 47 shows the results of quantitative PCR for factors involved inpluripotency and the undifferentiated cell state of naive fibroblasts(Naive), M-clusters (Cluster), and iPS cells (iPS). The meaning of eachcolumn is as defined in FIG. 46.

FIG. 48 shows the summary of a research report concerning the inductionefficiency of iPS cell lines prepared in human and mouse models. FIG. 48shows combinations of transcription factors inducing nuclearreprogramming.

FIGS. 49A, 49B and 49C show the results of FACS analysis usinganti-SSEA-3 antibody in human adipose tissue-derived mesenchymal stemcells. A: No stain, B: Second antibody only, C: SSEA-3 staining.

FIG. 50 shows the sequence of primers used for RT-PCR and positive andnegative control for each factor.

FIGS. 51A and 51B show the morphology of human subcutaneous adiposetissue (A) and the established mesenchymal cell derived from humansubcutaneous adipose tissue (B).

FIGS. 52A and 52B show the expression of SSEA-3 on commerciallyavailable human adipose-derived stem cell (HADSC) (A) and theestablished mesenchymal cell derived from human subcutaneous adiposetissue (B).

FIGS. 53A and 53B are photographs which show the morphology of Musecell-derived embryoid body (EB)-like cell cluster (M-cluster). FIG. 53Ashows the morphology of M-cluster obtained from commercially availablehuman adipose-derived stem cell (HADSC) and FIG. 53B shows themorphology of M-cluster obtained from the established mesenchymal cellderived from human subcutaneous adipose tissue.

FIG. 54 is the results of RT-PCT which shows the differentiation ofM-cluster. In the figure, “M-cluster I” indicates the differentiation ofM-cluster obtained from commercially available human adipose-derivedstem cell (HADSC) and “M-cluster II” indicates the differentiation ofM-cluster obtained from the subcutaneous adipose tissue-derivedmesenchymal cells.

FIGS. 55A, 55B and 55C are photographs of immunostaining which showsthat M-cluster formed from human adipose-derived stem cell (HADSC)differentiates into cells of three germ layers (tridermic cells). NF;neurofilament, ectodermal, CK7; cytokeratin7, endodermal, SMA; smoothmuscle actin, mesodermal.

FIG. 56 is a photograph which shows the expression of pluripotent cellmarkers of M-cluster formed from human adipose-derived stem cell(HADSC).

FIGS. 57A and 57B are photographs which show that Muse cell isnon-tumorigenic (4 months after transplantation) In contrast, mouse EScell formed teratoma by 8 weeks.

FIGS. 58A, 58B, 58C and 58D show photographs of HE staining of teratomagenerated from Mouse ES cells. In the figure, I is intestinal epithelium(endoderm) (FIG. 58B), II is neuronal element (ectoderm)(FIG. 58C) andIII is smooth muscle (mesoderm)(FIG. 58D).

FIGS. 59A and 59B are photographs of mouse testis to which Muse cellswere transplanted (4 months after transplantation). No sign of tumorformation was observed.

FIG. 60 is a diagram which shows human adipose-derived stem cell(HADSC)-derived Muse cell has self-renewal ability.

FIGS. 61A and 61B show the morphology of mesenchymal cells obtained fromhuman umbilical cord.

FIG. 62 shows the expression of SSEA-3 on mesenchymal cells obtainedfrom human umbilical cord. 1: No stain, 2: Second antibody only, 3:SSEA-3 staining.

FIG. 63 the morphology of M-cluster formed from SSEA-3 (+) cell isolatedfrom human umbilical cord.

FIG. 64 shows the results of the RT-PCR analysis for alpha-fetoprotein(α-FP; endoderm), GATA6 (endoderm), MAP-2 (ectderm) and Nkx2.5(mesoderm) of Muse cells isolated from umbilical cord.

FIG. 65 shows the expression of HLA class I antigen but not HLA class IIantigen of SSEA-3 positive cell which is derived from human bone marrowstroma cells.

FIG. 66 shows the expression of HLA class I antigen but not HLA class IIantigen of SSEA-3 positive cell which is derived from human dermalfibroblasts.

FIG. 67 shows the expression of HLA class I on Muse cells and non-Musecells.

FIG. 68 shows no expression of HLA class II on Muse cells nor non-Musecells.

FIG. 69 shows no non-specific reaction of Muse cells and non-Muse cellsto secondary antibody conjugated with Alexa568.

FIG. 70 shows the results of FACS analysis (CD14) of cells obtained fromhuman peripheral blood for lymphocyte stimulation test.

FIGS. 71A, 71B, 71C, 71D and 71E show the results of FACS analysis ofcells obtained by the induction of differentiation from monocytes tomonocyte-derived dendritic cell (MoDC) progenitor cells.

FIG. 72 shows the suppression of induction of differentiation frommonocytes to monocyte-derived dendritic cell (MoDC) progenitor cells inthe presence of Muse cells.

FIGS. 73A, 73B, 73C, 73D, 73E, 73F and 73G show the results of FACSanalysis of cells obtained by the induction of differentiation frommonocyte-derived dendritic cell (MoDC) progenitor cells to dendriticcells.

FIG. 74 shows the suppression of induction of differentiation frommonocyte-derived dendritic cell (MoDC) progenitor cells to dendriticcells in the presence of Muse cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be described in detail as follows.

The present invention relates to pluripotent stem cells or pluripotentstem cell fractions that can be directly obtained from body tissue of aliving body (in vivo), a method for isolating the pluripotent stem cellsor the pluripotent stem cell fractions, and the body tissue-derivedpluripotent stem cells or pluripotent stem cell fractions obtained bythe method. The pluripotent stem cells of the present invention arereferred to as Muse cells (multilineage-differentiating stress enduringcells).

In the present invention, the term “cell fraction” refers to a cellpopulation containing at least a given amount of a cell to be isolated.For example, the term “pluripotent stem cell fraction” refers to a cellpopulation containing a pluripotent stem cell in an amount correspondingto 1% or more thereof, 10% or more thereof, 30% or more thereof, 50% ormore thereof, 70% or more thereof, 90% or more thereof, or 95% or morethereof. Examples thereof include cell clusters obtained via culture ofpluripotent stem cells and cell populations obtained via enrichment ofpluripotent stem cells. Also, the cell fraction may also be referred toas a substantially homogenous cell fraction.

The term “living body” refers to a living mammalian body, and itspecifically refers to an animal body that undergoes development to someextent. In the present invention, examples of such living body do notinclude fertilized eggs or embryos at development stages before theblastula stage, but include embryos at development stages on and afterthe blastula stage, such as fetuses and blastulae. Examples of mammalsinclude, but are not limited to, primates such as humans and monkeys,rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep,pigs, cattle, horses, donkeys, goats, and ferrets. The pluripotent stemcells of the present invention are clearly distinguished from embryonicstem cells (ES cells) or embryonic germ stem cells (EG cells) in thatthey are from living body tissue.

The term “mesodermal tissue” refers to tissue of mesodermal origin thatappears in the course of initial development of an animal. Examples ofmesodermal tissue include tissue of the muscular system, connectivetissue, tissue of the circulatory system, tissue of the excretorysystem, and tissue of the genital system. For example, the pluripotentstem cells of the present invention can be obtained from bone marrowaspirates or skin tissue such as dermal connective tissue.

The term “mesenchymal tissue” refers to tissue such as bone, cartilage,fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon,dental pulp and umbilical cord. For example, the pluripotent stem cellsof the present invention can be obtained from the bone marrow or skin.Pluripotent cells derived from adipose tissue can be isolated fromadipose-derived stem cells (ADSCs) or adipose-dervied mesenchymal cellswhich are obtained from adipose tissue. Commercially available ADSCs canbe used. Adipose-derived stem cells are mesenchymal stem cells includedin fat tissue (adipose tissue). Adipose-derive stem cell fraction can beobtained from pannicular tissue or subcutaneous aspirate by the knownmethod. For example, mesenchymal cells (ADSC) established from adiposetissue according to Estes BT., et al. Nat Protoc.2010 Jul:5(7):1294-1311can be used. For cell surface antigens, adipose-derived stem cells areCD13 positive, CD29 positive, CD44 psitive, CD73 positive, CD90positive, CD105 positive, CD166 positive, CD14 negative, CD31 negativeand CD45 negative. Adipose-derived stem cells are commerciallyavailable. For example, human adipose-derived stem cells purachased fromLonza Group Ltd. Also, the pluripotent stem cells can be obtained fromthe umbilical cord. Unbilical cord is umbilical cord mesenchymal tissueof mammals. Umbilical cord is comprised of epidermis, vessel, blood, andmesenchymal tissue. Mesenchyaml tissue is origin of mesehcymal cellsfrom umbilical cord.

Both of Umbilical cord and fat tissue can be frozed for preservationbefore use.

The expression “cells can be directly obtained from tissue” means thatcells can be isolated from tissue without any artificial inductionoperation such as introduction of a foreign gene or a foreign protein ortreatment with a compound (e.g., administration of a compound). Suchforeign gene may be, but is not limited to, a gene capable ofreprogramming the nucleus of a somatic cell, for example. Examples ofsuch foreign gene include Oct family genes such as an Oct3/4 gene, Klffamily genes such as a Klf gene, Myc family genes such as a c-Myc gene,and Sox family genes such as a Sox2 gene. Also, examples of a foreignprotein include proteins encoded by these genes and cytokines.Furthermore, examples of a compound include a low-molecular-weightcompound capable of inducing the expression of the above gene that canreprogram the nucleus of a somatic cell, DMSO, a compound that canfunction as a reducing agent, and a DNA methylating agent. Thepluripotent stem cells of the present invention are clearlydistinguished from iPS cells (induced pluripotent stem cells) and EScells in that the pluripotent stem cells of the present invention can bedirectly obtained from living bodies or tissue. In addition, in thepresent invention, cell culture, isolation of a cell or a cell fractionusing a cell surface marker as an index, exposure of cells to cellularstress, and provision of a physical impact on cells are not included inexamples of artificial induction operation. Also, the pluripotent cellsof the present invention may also be characterized in that they can beobtained without requiring reprogramming or induction ofdedifferentiation.

The pluripotent stem cells of the present invention are thought to bepresent in mesodermal tissue or mesenchymal tissue, or the like of aliving body. In the present invention, cells or cell fractions existingin these types of tissue are isolated. The pluripotent stem cells of thepresent invention are present in the bone marrow, for example, so thatthey may be supplied from the bone marrow to each tissue of a livingbody via blood or the like. Hence, the pluripotent stem cells can beisolated from the bone marrow, each tissue of a living body, such asskin, and even blood.

The term “pluripotent stem cell(s)” refers to cells having pluripotencyand having the following properties.

-   (1) The pluripotent stem cells express pluripotency markers such as    Nanog, Oct3/4, SSEA-3, PAR-4, and Sox2.-   (2) The pluripotent stem cells exhibit clonality by which they    expand from a single cell and keep producing clones of themselves.-   (3) The pluripotent stem cells exhibit self-renewal capability.-   (4) The pluripotent stem cells can differentiate in vitro and in    vivo into the three germ layers (endodermal cell lineage, mesodermal    cell lineage, and ectodermal cell lineage).-   (5) The pluripotent stem cells differentiate into the three germ    layers when transplanted into the testis or subcutaneous tissue of a    mouse.-   (6) The pluripotent stem cells are found to be positive through    alkaline phosphatase staining.

The pluripotent stem cells of the present invention are clearlydistinguished from adult stem cells and tissue stem cells in thatpluripotent stem cells of the present invention have pluripotency. Also,the pluripotent stem cells of the present invention are clearlydistinguished from cell fractions such as bone marrow stromal cells(MSC) in that pluripotent stem cells of the present invention areisolated in the form of a single cell or a plurality of cells havingpluripotency.

Moreover, the pluripotent stem cells of the present invention have thefollowing properties.

-   (i) The growth rate is relatively gentle and the division cycle    takes 1 day or more, such as 1.2-1.5 days. However, the pluripotent    stem cells do not exert infinite proliferation in a manner similar    to ES cells or iPS cells.-   (ii) When transplanted into an immunodeficient mouse, the    pluripotent stem cells differentiate into an endodermal cell    lineage, a mesodermal cell lineage, and an ectodermal cell lineage.    The pluripotent stem cells are characterized in that they do not    become tumorigenic cells for a half year or longer, unlike ES cells    or iPS cells, whereby teratomas are formed within a short time    period such as 8 weeks.-   (iii) The pluripotent stem cells form embryoid body-like cell    clusters as a result of suspension culture.-   (iv) The pluripotent stem cells form embryoid body-like cell    clusters as a result of suspension culture and stop growth within    about 10-14 days. Subsequently, when the clusters are transferred    for adherent culture, they start to grow again.-   (v) Asymmetric division is associated with growth.-   (vi) The karyotypes of the cells are normal.-   (vii) The pluripotent stem cells have no or low telomerase activity.    The expression “. . . have no or low telomerase activity” refers to    no or low telomerase activity being detected when such activity is    detected using a TRAPEZE XL telomerase detection kit (Millipore),    for example. The term “low telomerase activity” refers to a    situation in which cells have telomerase activity to the same degree    as that of human fibroblasts or have telomerase activity that is 1/5    or less and preferably 1/10 or less that of Hela cells.-   (viii) Regarding methylation state, methylation levels in Nanog and    Oct3/4 promoter regions are low in iPS cells induced from Muse    cells.-   (ix) The pluripotent stem cells exhibit high phagocytic ability.-   (x) The pluripotent stem cells exhibit no tumorigenic proliferation.    Here, the expression “...cells exhibit no tumorigenic proliferation”    refers to a situation in which, when suspension culture is    performed, the cells stop their growth at the time when their    clusters reach a predetermined size and do not undergo infinite    growth. Moreover, such expression refers to a situation in which,    when such cells are transplanted into the testis of an    immunodeficient mouse, no teratoma is formed. In addition, the    above (i) to (iv) and the like also relate to the fact that the    relevant cells (clusters) do not undergo tumorigenic proliferation.

Specifically, the cells of the present invention are the followingpluripotent stem cells, for example:

-   (A) pluripotent stem cells that are obtained from mesodermal tissue,    mesenchymal tissue, or the like of a living body and can be directly    obtained without introduction of a chemical substance, a foreign    gene, or a foreign protein into such cells;-   (B) pluripotent stem cells having the property of (1) above, wherein    mesodermal tissue or mesenchymal tissue of a living body is selected    from the group consisting of bone marrow, skin, blood, umbilical    cord, and fat;-   (C) the pluripotent stem cells of (A) or (B) above that can be    obtained without reprogramming or induction of dedifferentiation;-   (D) the pluripotent stem cells of (A) or (B) above that do not    become tumorigenic at least within half a year after being    transplanted into the testis;-   (E) the pluripotent stem cells of (A) or (B) above that do not    undergo infinite growth, unlike ES cells and iPS cells; or-   (F) pluripotent stem cells from mesodermal tissue or mesenchymal    tissue of a living body, which survive when treated with protease    and thus are resistant to protease.

Moreover, the pluripotent stem cells of the present invention can beenriched by placing cellular stress on the cells of mesodermal tissue ormesenchymal tissue of a living body and then collecting surviving cells.Here, the term “cellular stress” refers to external stress.Specifically, cells are exposed to such stress via protease treatment,culture under low-oxygen conditions, culture under low phosphateconditions, culture under serum starvation conditions, culture in asugar starvation state, culture under exposure to radiation, cultureunder exposure to heat shock, culture in the presence of a toxicsubstance, culture in the presence of active oxygen, culture undermechanical stimulation, culture under pressure treatment, or the like.Of these examples, protease treatment, and specifically, culture in thepresence of protease, is preferred. Protease is not limited. Serineprotease such as trypsin and chymotrypsin, aspartic protease such aspepsin, cysteine protease such as papain and chymopapain,metalloprotease such as thermolysin, glutamic protease, N-terminalthreonine protease, and the like can be used. The concentration ofprotease to be added for culture is not limited. In general,concentrations to be employed for removal of adherent cells that arecultured in petri dishes or the like may be employed herein. Thepluripotent stem cells of the present invention can be said to be sterncells having resistance to the above-mentioned external stresses, suchas cells having resistance to trypsin.

Examples of mesodermal tissue and mesenchymal tissue of a living bodyinclude, but are not limited to, bone-marrow mononuclear cells,fibroblast fractions such as skin cells, pulp tissue, eyeball tissue,and hair root tissue. As cells, both cultured cells and cells collectedfrom tissue can be used. Among these cells, bone marrow cells and skincells are desired. Examples of such cells include a human bone marrowstromal cell (MSC) fraction and a human dermal fibroblast fraction. Abone marrow stromal cell (MSC) fraction can be obtained by culturing abone marrow aspirate for 2 to 3 weeks.

Most cells of tissue subjected to the various above stresses will die.Surviving cells include the pluripotent stem cells of the presentinvention. After stress is placed on cells, dead cells should beremoved. However, when protease is used, these dead cells are lysed viathe effects of protease.

Also, after stress is placed on cells, a physical impact is provided tothe cells to make dead or dying cells become easily disrupted, and thenthose cells may be removed. A physical impact can be provided byrigorous pipetting, rigorous stirring, vortexing, or the like.

Cellular stress is placed on cells, a physical impact is provided ifnecessary, and then the resulting cell populations are subjected tocentrifugation. The resulting surviving cells are obtained and collectedas pellets, so that the pluripotent stem cells of the present inventioncan be isolated. Also, from the thus obtained cells, the pluripotentstem cells or pluripotent cell fractions of the present invention can beisolated using the following surface markers as indices.

The pluripotent stem cells or pluripotent cell fractions of the presentinvention can also be enriched by culturing mesodermal tissue,mesenchymal tissue, or the like (in vivo) of a body subjected to stresssuch as trauma or a burn and then collecting cells that have migrated.Cells of damaged tissue are exposed to stress. Hence, in the presentinvention, the expression “culture of mesodermal tissue or mesenchymaltissue (in vivo) of a damaged body” also refers to placing cellularstress on cells of mesodermal tissue, mesenchymal tissue, or the like ofa living body.

As an example, a method for treating such cells with trypsin is asdescribed below. The concentration of trypsin at this time is notlimited. For example, in general culture of adherent cells, theconcentrations of trypsin may be concentrations that are employed forremoval of adherent cells adhering to a culture vessel, ranging from0.1% to 1% and preferably ranging from 0.1% to 0.5%, for example. Forexample, cells can be exposed to external stress by incubating cells(100,000-500,000 cells) from mesodermal tissue, mesenchymal tissue, orthe like of a living body in 5 ml of a trypsin solution with the aboveconcentration. The time for trypsin incubation ranges from about 5 to 24hours and preferably ranges from about 5 to 20 hours. In the presentinvention, 8 or more hours of trypsin incubation, such as 8 hours or 16hours of treatment, is long-term trypsin incubation.

After trypsin incubation, a physical impact is desirably provided bypipetting, stirring, vortexing, or the like, as described above. This isperformed to remove dead cells or dying cells.

When suspension culture is performed after trypsin incubation,incubation is desirably performed in gel such as methylcellulose gel, inorder to prevent cell-to-cell aggregation. Also, a cell culture vesselis desirably coated in advance with poly(2-hydroxyethyl methacrylate) orthe like in order to prevent adhesion of cells to the culture vessel andmaintain the state of suspension.

When cells exposed to external stress, collected by centrifugation, andthen cultured, cells form cell clusters. The size of such a cell clusterranges in diameter from about 25 μm to 150 μm. The pluripotent stemcells (Muse cells) of the present invention are included in an enrichedstate within a cell fraction that has survived after exposure toexternal stress. Such cell fraction is referred to as Muse-enriched cellfractions (Muse enriched populations). The percentage of Muse cells insuch a Muse-enriched cell fraction differs depending on method of stresstreatment.

The fact that the pluripotent stem cells or the pluripotent stem cellfractions of the present invention survive after exposure to stresssuggests that the pluripotent stem cells or the pluripotent stem cellfractions of the present invention are resistant to such stress.

Regarding the medium to be used for culturing cells from mesodermaltissue, mesenchymal tissue, or the like of a living body and cultureconditions, any medium and culture conditions generally used forculturing animal cells may be employed. Also, a known medium forculturing stem cells may be used. A medium may be appropriatelysupplemented with serum such as fetal calf serum, antibiotics such aspenicillin and streptomycin, and various bioactive substances.

Furthermore, the present invention also encompasses pluripotent stemcells which are derived cells or induced cells of the pluripotent stemcells of the present invention that can be directly obtained from themesodermal tissue, mesenchymal tissue, or the like of a living body. Theterm “derived cells or induced cells” refers to cells or cell fractionsobtained by culturing the pluripotent stem cells or cells obtained bysubjecting the pluripotent stem cells to an artificial inductionoperation such as introduction of a foreign gene. Progeny cells are alsoincluded herein. In addition, it is said that iPS cells that had beenreported at the time of the present invention are induced frompluripotent stem cells as a result of reprogramming (e.g., introductionof foreign genes into somatic cells, such as dermal fibroblasts). Cellsobtained by subjecting cells of the present invention (that can bedirectly obtained from the tissue of the present invention and alreadyhave properties as pluripotent stem cells) to an artificial inductionoperation such as introduction of a foreign gene are distinguished fromiPS cells.

Embryoid body-like (EB body-like) cell clusters are obtained throughsuspension culture of the pluripotent stem cells of the presentinvention. The present invention also encompasses such embryoidbody-like cell clusters and cells contained in such embryoid body-likecell clusters. Embryoid bodies are formed as cell clusters throughsuspension culture of the pluripotent stem cells such as ES cells. Atthis time, in the present invention, such an embryoid body-like cellclusters obtained by culturing the pluripotent stem cells of the presentinvention is also referred to as a M-cluster (Muse cell-derived embryoidbody-like cell cluster). Examples of a method for suspension culture forthe formation of embryoid body-like cell clusters include culture usingmedium containing a water soluble polymer such as methylcellulose(Nakahata, T. et al., Blood 60, 352-361 (1982)) and hanging drop culture(Keller, J. Physiol. (Lond) 168: 131-139, 1998). The present inventionalso encompasses embryoid body-like cell clusters obtained viaself-renewal from the embryoid body-like cell clusters, cells containedin such embryoid body-like cell clusters, and pluripotent stem cells.Here, the term “self-renewal” refers to a situation in which cellscontained in embryoid body-like cell clusters are cultured so as tocause the formation of embryoid body-like cell clusters again.Self-renewal may be performed by repeating a cycle once to severalinstances. Also, the present invention also encompasses cells andtissue, which differentiate from either the above embryoid body-likecell clusters or cells contained in such embryoid body-like cellclusters.

FIG. 1 shows the relationship among mesenchymal cell (human fibroblast,human bone marrow stromal cell (MSC), and fresh bone marrow fluid)fractions, Muse cells, and M-clusters. When stress stimulation (e.g.,long-term trypsin incubation (LTT)) is imposed upon mesenchymalcell-like cell clusters, Muse cells are enriched, and then cellfractions containing many Muse cells (referred to as a Muse-enrichedcell fraction) are obtained. Through suspension culture of Muse cells inthe cell fraction, an embryoid body-like cell cluster (M-cluster) isobtained. When embryoid body-like cell clusters are cultured in aculture dish coated with gelatin, cells differentiate into cells of the3 germ layers. Also, as shown in FIG. 1, SSEA-3 (+) cells are directlyseparated and then suspension culture is performed without exposingcells to long-term stress, so that M-clusters can be obtained.

When the growth of Muse cells is stopped once via suspension culture,Muse cells initiate growth when transferred for adherent culture.Through repetition of separation using suspension culture-adherentculture-SSEA-3 expression as an index, Muse cells can be grown in largeamounts (FIG. 2).

Furthermore, the pluripotent stem cells or pluripotent cell fractions ofthe present invention can also be directly isolated from body tissuewithout exposure to cellular stress. Specifically, the pluripotent stemcells or the pluripotent stem cell fractions of the present inventioncan be isolated from mesodermal tissue, mesenchymal tissue, or the likeof a living body by the following method without an induction operationsuch as introduction of a foreign gene.

Examples of body tissue include, but are not limited to, mesodermaltissue and mesenchymal tissue of a living body such as bone marrow,skin, and umbilical cord tissue. When bone marrow is used, a mononuclearcell fraction of the bone marrow can be used. Isolation can be performedusing a cell surface marker that is expressed richly on the surface ofMuse cells. For example, isolation can be performed using SSEA-3expression as an index. The pluripotent stem cells of the presentinvention may also be referred to as SSEA-3 (+) Muse cells. Moreover,Muse cells express CD105, which is a mesenchymal marker. Muse cells arepositive positive for SSEA-3, and positive for CD105. Therefore, Musecells can be isolated using the expression of both SSEA-3 and CD105 asan index. With the use of these cell surface markers, the pluripotentstem cells of the present invention can be isolated in the form ofsingle cells. The thus isolated single cells can be grown by culture. Inaddition, the present invention encompasses pluripotent stem cells thatcan be isolated from body tissue of a mammal other than a human using amarker SSEA-3 or markers corresponding to SSEA-3. For example, 40 to 70%of cells isolated from HADSC using SSEA-3 as a maker are Muse cellswhich can form M-cluster.

Meanwhile, Muse cells are negative for NG2, CD34, vWF (von Willebrandfactor), c-kit (CD117), CD146, and CD271 (NGFR). Moreover, Muse cellsare negative for Sox10, Snai1, Slug, Tyrp1, and Dct.

Whether or not cells are negative for surface antigens such as NG2,CD34, vWF, CD117, CD146, and CD271 or whether or not the expressionthereof is weak can be determined by microscopically observing whetheror not cells are stained with antibodies (against these antigens)labeled with a chromogenic enzyme, a fluorescent compound, or the like.For example, cells are immunostained with these antibodies, so that thepresence or the absence of a surface antigen can be determined. Thepresence or the absence of the same can also be determined usingantibody-conjugated magnetic beads. Also, the presence or the absence ofa surface antigen can be determined using FACS or a flowcyte meter. As aflowcyte meter, FACSAria (Becton Dickinson), FACS vantage (BectonDickinson), FACS Calibur (Becton Dickinson), MACS (magnetic cellsorting) or the like can be used, for example.

Regarding transcription factors such as Sox10, Snai1, Slug, Tyrp1, andDct, the expression thereof can also be examined by a technique such asRT-PCR.

The expression “. . . are negative for these surface antigens” refers tothat a situation in which, when FACS analysis is conducted as describedabove, cells are not sorted as positive cells or when expression isexamined by RT-PCR, no expression thereof is confirmed. Even if suchsurface antigens are expressed to a degree such that they areundetectable by such techniques, cells are designated as negative in thepresent invention. Also, at the same time, measurement is performed withcells such as hematopoietic stem cells known to be positive for theabove markers. When almost no expression is detected or the expressionlevel is significantly lower compared with such positive cells, cellsmay be designated as negative.

Cells of the present invention can be isolated based on the propertiesof the aforementioned cell surface antigens.

As described above, Muse cells can be isolated using “being positive forSSEA-3” as an index. Moreover, Muse cells can be isolated using theexpression of CD105 as an index. Muse cells can be further isolatedusing non-expression of at least 1, such as 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11, markers selected from the group consisting of NG2, CD34, vWF (vonWillebrand factor), c-kit (CD117), CD146, CD271 (NGFR), Sox10, Snai1,Slug, Tyrp1, and Dct, as an index. For example, isolation is possibleusing non-expression of CD117 and CD146. Furthermore, isolation can beperformed using non-expression of CD117, CD146, NG2, CD34, vWF, andCD271 as an index. Furthermore, isolation can be performed usingnon-expression of the above 11 markers as an index.

When isolation is performed using a surface marker(s), 1 or a pluralityof pluripotent stern cells of the present invention can be directlyisolated from mesodermal tissue, mesenchymal tissue, or the like of aliving body without culture or the like. Also, the pluripotent stemcells of the present invention can be identified and isolated byvisually observing the cell morphology using a microscope or the like.

After provision of a cellular stress to mesodermal tissue, mesenchymaltissue, or the like of a living body, isolation may also be performedfrom a surviving cell group using a surface marker.

Also, the pluripotent stem cells or the pluripotent cell fractions ofthe present invention can be characterized by high-level expression ofanother specific factor, in addition to the use of the above markers.

Muse cells that are the pluripotent stem cells of the present inventioncan be obtained from naive bone marrow stromal cell (MSC) fractions ordermal fibroblast fractions. Muse cells are further cultured, so thatMuse cell-derived embryoid body (EB)-like cell clusters (M-clusters) areobtained. Through comparison and examination of factors expressed inMuse cells, naive cells, Muse-derived embryoid body-like cell clusters,and human ES cells, a factor expressed at high levels in Muse cells canbe detected. Examples of such factors include gene transcriptionproducts, proteins, lipids, and saccharides.

FIG. 3 shows factors for which the ratio of the expression level inM-clusters to the same in naive cells is high. In particular, the ratiois high for the following 18 factors.

-   (i) SSEA-3-   (ii) v-fos FBJ murine osteosarcoma viral oncogene homolog-   (iii) solute carrier family 16, member 6 (monocarboxylic acid    transporter 7)-   (iv) tyrosinase-related protein 1-   (v) Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit-   (vi) chromosome 16 open reading frame 81-   (vii) chitinase 3-like 1 (cartilage glycoprotein-39)-   (viii) protease, serine, 35-   (ix) kynureninase (L-kynurenine hydrolase)-   (x) solute carrier family 16, member 6 (monocarboxylic acid    transporter 7)-   (xi) apolipoprotein E-   (xii) synaptotagmin-like 5-   (xiii) chitinase 3-like 1 (cartilage glycoprotein-39)-   (xiv) ATP-binding cassette, sub-family A (ABC1), member 13-   (xv) angiopoietin-like 4-   (xvi) prostaglandin-endoperoxide synthase 2 (prostaglandin G/H    synthase and cyclooxygenase)-   (xvii) stanniocalcin 1-   (xviii) coiled-coil domain containing 102B

The pluripotent stem cells or the pluripotent stem cell fractions of thepresent invention are characterized in that at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 factors above are expressedat high levels. Hence, the pluripotent stem cells or the pluripotentstem cell fractions can be isolated using high-level expression of atleast 2 factors as an index.

FIG. 4 shows factors for which the ratio of the expression level inM-clusters to the same in human ES cells is high. In particular, theratio is high in the following 20 factors.

-   (a) matrix metallopeptidase 1 (interstitial collagenase)-   (b) epiregulin-   (c) chitinase 3-like 1 (cartilage glycoprotein-39)-   (d) Transcribed locus-   (e) chitinase 3-like 1 (cartilage glycoprotein-39)-   (f) serglycin-   (g) MRNA full length insert cDNA clone EUROIMAGE 1913076-   (h) Ras and Rab interactor 2-   (i) lumican-   (j) CLCA family member 2, chloride channel regulator-   (k) interleukin 8-   (l) Similar to LOC166075-   (m) dermatopontin-   (n) EGF, latrophilin and seven transmembrane domain containing 1-   (o) insulin-like growth factor binding protein 1-   (p) solute carrier family 16, member 4 (monocarboxylic acid    transporter 5)-   (q) serglycin-   (r) gremlin 2, cysteine knot superfamily, homolog (Xenopus laevis)-   (s) insulin-like growth factor binding protein 5-   (t) sulfide quinone reductase-like (yeast)

The pluripotent stem cells or the pluripotent stem cell fractions of thepresent invention are characterized in that at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 factors above areexpressed at high levels. Hence, the pluripotent stem cells or thepluripotent stem cell fractions can be isolated using high-levelexpression of at least 2 factors as an index.

Furthermore, in the pluripotent stem cells or the pluripotent stem cellfractions of the present invention, at least 2 of the above factors(i)-(xviii) and at least 2 of the above factors (a)-(t) may besimultaneously expressed at high levels. Hence, the pluripotent stemcells or the pluripotent stem cell fractions can be isolated usinghigh-level expression of these genes as an index.

Furthermore, the pluripotent stem cells or the pluripotent stem cellfractions of the present invention are characterized in that: factors ofan odorant receptor (olfactory receptor) group and factors of achemokine receptor group, other than pluripotency markers, areexpressed; that is, they are positive for specific odorant receptors orchemokine receptors.

Examples of odorant receptors that are expressed in the pluripotent stemcells or the pluripotent stem cell fractions of the present inventioninclude the following 22 receptors.

-   olfactory receptor, family 8, subfamily G, member 2 (OR8G2);-   olfactory receptor, family 7, subfamily G, member 3 (OR7G3);-   olfactory receptor, family 4, subfamily D, member 5 (OR4D5);-   olfactory receptor, family 5, subfamily AP, member 2 (OR5AP2);-   olfactory receptor, family 10, subfamily H, member 4 (OR1OH4);-   olfactory receptor, family 10, subfamily T, member 2 (OR10T2);-   olfactory receptor, family 2, subfamily M, member 2 (OR2M2);-   olfactory receptor, family 2, subfamily T, member 5 (OR2T5);-   olfactory receptor, family 7, subfamily D, member 4 (OR7D4);-   olfactory receptor, family 1, subfamily L, member 3 (OR1L3);-   olfactory receptor, family 4, subfamily N, member 4 (OR4N4);-   olfactory receptor, family 2, subfamily A, member 7 (OR2A7);-   guanine nucleotide binding protein (G protein), alpha activating    activity polypeptide, olfactory type (GNAL);-   olfactory receptor, family 6, subfamily A, member 2 (OR6A2);-   olfactory receptor, family 2, subfamily B, member 6 (OR2B6);-   olfactory receptor, family 2, subfamily C, member 1 (OR2C1);-   olfactory receptor, family 52, subfamily A, member I (OR52A1);-   olfactory receptor, family 10, subfamily H, member 3 (OR1OH3);-   olfactory receptor, family 10, subfamily H, member 2 (ORIOH2);-   olfactory receptor, family 51, subfamily E, member 2 (OR51E2);-   olfactory receptor, family 5, subfamily P, member 2 (OR5P2); and-   olfactory receptor, family 10, subfamily P, member 1 (OR10P1)

Examples of a chemokine receptor that is expressed in the pluripotentstem cells or pluripotent stem cell fractions of the present inventioninclude the 5 following receptors.

-   chemokine (C-C motif) receptor 5 (CCRS);-   chemokine (C-X-C motif) receptor 4 (CXCR4);-   chemokine (C-C motif) receptor 1 (CCR1);-   Duffy blood group, chemokine receptor (DARC); and-   chemokine (C-X-C motif) receptor 7 (CXCR7).

The pluripotent stem cells or the pluripotent stem cell fractions of thepresent invention express at least one of the above olfactory receptorsor express at least one of the above chemokine receptors.

Because of the effects of these odorant receptors or chemokine receptorsand migratory factors that bind to the receptors, the pluripotent stemcells of the present invention migrate to damaged tissue and thensurvive and differentiate at the tissue. For example, when the liver,skin, spinal cord, or muscle is damaged, a specific migratory factor andan odorant receptor expressed on the cell surfaces function to cause thepluripotent stem cells to migrate to the relevant tissue, survive at thetissue, and then differentiate into liver (endoderm), skin (ectoderm),spinal cord (ectoderm), or muscle (mesoderm) cells, so that the tissuecan be regenerated.

In a Muse-enriched cell fraction richly containing Muse cells that arethe pluripotent stem cells of the present invention, Rex1, Sox2, KLF-4,c-Myc, DPPA2, ERAS, GRB7, SPAG9, TDGF1, and the like are upregulated. Ina cell cluster of Muse cells, DAZL, DDX4, DPPA4, Stella, Hoxb 1, PRDM1,SPRY2, and the like are upregulated.

Also, in pluripotent stem cells or the pluripotent stem cell fractionsof the present invention, the expression of CD34 and CD117 hematopoieticstem cell markers is never observed or is observed at an extremely lowlevel.

The present invention encompasses not only Muse cells, but also a cellfraction resulting from enrichment of Muse cells, a cell fractionresulting from growth of Muse cells, and a cell fraction resulting fromdifferentiation of Muse cells. The present invention further encompassesa research kit, a cell chip, and a therapeutic device containing Musecells or cells derived from Muse cells.

The pluripotent stem cells of the present invention have pluripotencyand thus are able to differentiate into all types of tissue. Thepluripotent stem cells or the pluripotent cell fractions can be used forregeneration medicine and the like. For example, such cells and cellfractions can be used for regeneration of various types of tissue,various organs, and the like. Specific examples thereof include skin,cerebro-spinal cord, liver, and muscle. The pluripotent stem cells orthe pluripotent stem cell fractions of the present invention areadministered directly to or to an area in the vicinity of injured ordamaged tissue, organs, and the like, so that the pluripotent stem cellsenter the tissue or organ and differentiate into cells unique to therelevant tissue or organ. In this manner, the pluripotent stem cells cancontribute to the regeneration or reconstruction of tissue and organs.Also, the systemic administration of the pluripotent stem cells orpluripotent stem cell fractions is possible by intravenousadministration or the like. In this case, the pluripotent stem cells aredirected by homing or the like to a damaged tissue or organ, reach andenter the tissue or organ, and then differentiate into cells of thetissue or organ, so as to be able to contribute to tissue or organregeneration and reconstruction.

Administration can be performed via parenteral administration such assubcutaneous injection, intravenous injection, intramuscular injection,and intraperitoneal injection, oral administration, or intrauterineinjection into an embryo, for example. Also, local administration orsystemic administration may be performed herein. Local administrationcan be performed using a catheter, for example. The dose can beappropriately determined depending on an organ to be regenerated, atissue type, or a size.

Examples of an organ to be regenerated include, but are not limited to,bone marrow, spinal cord, blood, spleen, liver, lungs, bowel, eyes,brain, immune system, circulatory system, bone, connective tissue,muscle, heart, blood vessel, pancreas, central nervous system,peripheral nervous system, kidney, bladder, skin, epithelial appendages,breast-mammary gland, adipose tissue, and mucous membranes of mouth,esophagus, vagina, and anus, for example. Also, examples of diseases tobe treated therein include, cancer, cardiovascular disease, metabolicdisease, hepatic disease, diabetes mellitus, hepatitis, haemophilia,blood system disease, degenerative or traumatic neurologic disorder suchas spinal cord injury, autoimmune disease, genetic defects, connectivetissue disease, anemia, infectious disease, graft rejection, ischaemia,inflammation, and damage to skin or muscle.

Cells may be administered with a pharmaceutically acceptable basematerial. Such base material may be made of a substance with highbio-compatibility, such as collagen or a biodegradable substance. Theymay be in the form of particles, plates, tubes, vessels, or the like.Cells may be administered after binding thereof to a base material orafter causing a base material to contain cells therein.

Also, in vitro differentiation induction is performed for pluripotentstem cells of the present invention, tissue is constructed using cellsthat have further differentiated, and then the differentiated cells ortissue may be transplanted. Since the pluripotent stem cells of thepresent invention do not undergo tumorigenic transformation, aprobability of tumor formation of the cells is low and can be said to besafe, even when the undifferentiated pluripotent stem cells of thepresent invention are contained in the above transplanted differentiatedcells or tissue. To prevent rejection of transplanted cells or tissue bya recipient in such regeneration medicine, it is desired that mesodermaltissue, mesenchymal tissue, or the like is collected from a patient tobe subjected to regeneration medicine, and then pluripotent stem cellsor pluripotent cell fractions of the present invention are isolated fromthe relevant tissue for use. Furthermore, the pluripotent stem cells orthe pluripotent stem cell fractions of the present invention can be usedfor treatment of diseases due to tissue degeneration or dysfunction. Inthis case, for example, the pluripotent stem cells or the pluripotentstem cell fractions of the present invention are enriched ex vivo,grown, or caused to differentiate and then returned into the body. Forexample, the pluripotent stem cells are caused to differentiate intospecific tissue cells and then the cells are transplanted into tissue tobe treated. Also, in situ cell therapy can be performed bytransplantation of such cells. In this case, examples of target cellsinclude hepatic cells, neural cells such as neuronal cells or glialcells, skin cells, and muscle cells such as skeletal muscle cells. Thepluripotent stem cells of the present invention are caused todifferentiate into these cells, the differentiated cells aretransplanted, and then treatment can be performed in situ. Through suchtreatment, Parkinson's disease, brain infarction, spinal cord injury,myodystrophy, and the like can be treated, for example. Since thepluripotent stem cells of the present invention do not undergotumorigenic transformation, they unlikely become tumors and safe even ifused for such treatment.

Also, the pluripotent stem cells of the present invention are caused todifferentiate to form blood or blood components, so that blood or bloodcomponents can be formed ex vivo or in vitro. Examples of such bloodcomponents include erythrocytes, leukocytes, and blood platelets. Thethus formed blood or blood components can be used for autologoustransfusion or cross transfusion.

As described above, when the pluripotent stem cells or the pluripotentstem cell fractions of the present invention are used for treatment,their differentiation may be caused ex vivo, in vivo, or in vitro. Thepluripotent stem cells of the present invention differentiate intoosteoblasts, chondrocytes, adipocyte, fibroblasts, bone-marrow stroma,skeletal muscle, smooth muscle, myocardium, eyes, endothelium,epithelium, liver, pancreas, hematopoietic system, glia, neuronal cells,or oligodendroglial cell, for example. Differentiation of thepluripotent stem cells of the present invention can be achieved byculturing them in the presence of a differentiation factor. Examples ofa differentiation factor include a basic fibroblast growth factor(bFGF), a vascular endothelium growth factor (VEGF), a dimethylsulfoxide (DMSO), and isoproterenol; or a fibroblast growth factor 4(FGF4) and a hepatocyte growth factor (HGF). The present invention alsoencompasses cells that have differentiated from the pluripotent stemcells of the present invention.

When the pluripotent stem cells of the present invention are used fortreatment, a gene encoding a protein antitumor substance, a bioactivesubstance, or the like may be introduced. Therefore, it can be said thatthe pluripotent stem cells of the present invention have a function forthe delivery of a therapeutic agent. Examples of such substance includeantiangiogenic agents.

The present invention encompasses materials for cell transplantationtherapy or compositions for cell transplantation therapy, or materialsfor regeneration medicine or compositions for regeneration medicine,which contain Muse cells, embryoid body-like cell clusters formed ofMuse cells, and cells or tissue/organs obtained via differentiation fromMuse cells or the above embryoid body-like cell clusters. Such acomposition contains a pharmaceutically acceptable buffer, diluent, orthe like in addition to Muse cells, an embryoid body-like cell clusterformed of Muse cells, or cells or tissue and/or organ obtained throughdifferentiation from Muse cells or the above embryoid body-like cellcluster.

Moreover, cells are collected from a patient, Muse cells are isolated,and then the Muse cells can be used for various diagnoses. For example,a patient's genes are collected from Muse cells and then the geneinformation is obtained, so that precise diagnosis reflecting theinformation becomes possible. For example, cells of each tissue and/ororgan having the same characteristics (e.g., genetic background) asthose of a subject can be obtained by causing differentiation ofpatient's cell-derived Muse cells. Hence, regarding disease diagnosis,elucidation of pathological conditions, diagnosis for the effects oradverse reactions of drugs, or the like, appropriate diagnosis can bemade according to the characteristics of each subject. Specifically,Muse cells, embryoid body-like cell clusters formed of Muse cells, andcells or tissue and/or organs obtained through differentiation of Musecells or the above embryoid body-like cell clusters can be used asdiagnostic materials. For example, the present invention encompasses amethod for diagnosing the disease or the like of a subject using Musecells isolated from the subject or using tissue or an organ (obtainedvia differentiation from the Muse cells) having the same geneticbackground as that of the subject.

Also, somatic cells can be obtained in large amounts via differentiationof Muse cells. Hence, basic research such as elucidation of a diseasemechanism, development of a therapeutic agent, screening for the effectsof a drug or toxicity, drug evaluation, and the like can be performed.Specifically, Muse cells, embryoid body-like cell clusters formed ofMuse cells, and cells or tissue and/or organs obtained throughdifferentiation of Muse cells or the above embryoid body-like cellclusters can be used as materials for drug evaluation or drug screening.For example, the present invention encompasses a method for screeningfor a drug or evaluating a drug, comprising causing differentiationand/or growth of Muse cells, obtaining somatic cells, administering acandidate drug to the somatic cells, and then examining the response ofsomatic cells.

Also, a Muse cell bank is constructed by constructing a library ofvarious (e.g., various types of HLA) Muse cells, so that a systemcapable of providing Muse cells to Muse cell application sites accordingto need can be realized. For example, in addition to the above listedpurposes, provision of cells with no (or little) rejections to urgentlyrequired cell transplantation therapy can be performed. Specifically,the present invention encompasses a method for constructing a Muse celllibrary; that is, a Muse cell bank, having different genetic properties,comprising isolating and collecting Muse cells having various geneticproperties. Also, a library or a bank can also be constructed using notonly Muse cells, but also an embryoid body-like cell cluster formed fromMuse cells, and cells or tissue and/or organ obtained throughdifferentiation from Muse cells or the above embryoid body-like cellclusters. In the present invention, libraries or banks that areconstructed by obtaining embryoid body-like cell clusters formed ofthese Muse cells, and cells or tissue and/or organs obtained throughdifferentiation of Muse cells or the above embryoid body-like cellclusters are also referred to as cell libraries or cell banks. Thepresent invention encompasses the thus constructed cell libraries orcell banks. Such cell libraries or cell banks comprise vessels such as aplurality of tubes containing cells and the like having differentgenetic characteristics. Such cells may also be frozen. For example,when tissue or an organ is transplanted into a subject or regenerationthereof is required, cells appropriate in terms of genetic background orthe like of the subject are selected from the above cell library or cellbank. Thus, transplantation or regeneration therapy can be performedusing the cells.

The present invention encompasses a therapeutic method, comprisingadministering, for treatment of a disease, a therapeutically effectivedose of the pluripotent stem cells, a cell fraction thereof of thepresent invention, or cells derived or induced from such cells to apatient who needs treatment. The effective dose can be specified basedon the number of cells to be administered, for example, andappropriately determined depending on disease types or severity. In theabove therapeutic method, the pluripotent stern cells of the presentinvention do not form any teratoma, so that no tumor is formed in apatient. Also, when autologous cell-derived Muse cells are administered,there is no need to cause bone marrow dysfunction by subjecting apatient to radiation exposure, chemotherapy, or the like. When Musecells that are not autologous cells are used, the above treatment isperformed.

Furthermore, Muse cells can be a source of iPS cells (inducedpluripotent stem cells). Efficiency for preparation of iPS cells usingMuse cells as a source is much higher (at least higher by 25 or morefolds) than that of a case of using another type of cells (e.g., dermalfibroblasts not fractioned using SSEA-3 expression as an index) as asource.

iPS cells can be prepared by introducing a specific gene or a specificcompound into Muse cells so as to alter cytoplasms. Alterations ofcytoplasms include reprogramming or tumorigenesis, for which currentlyknown methods or all methods that will be established in the future canbe employed.

For example, a gene is introduced into Muse cells according to thedescription of JP Patent No. 4182742 or the description in FIG. 48, sothat iPS cells can be established from Muse cells. Also, in addition tothe method described in FIG. 48, it can be said that iPS cells can beestablished through introduction of a chemical substance, a foreigngene, or a foreign protein. Establishment of iPS cells from Muse cellscan be performed by methods described in Examples described later, forexample.

The iPS cells obtained as described above from Muse cells may also bereferred to as “Muse-derived iPS cells (Muse-iPSC).” The presentinvention encompasses such Muse-derived iPS cells. Muse-derived iPScells can be said to be pluripotent stem cells which were derived fromMuse cell-derived and had proliferative ability.

The present invention further encompasses a cell therapy composition forallotransplantation which comprises SSEA-3 positive pluripotent stemcell derived from body tissue, which are Muse cells.

Alloplantation means the plantation of cell or tissue of another person(another individual).

Muse cells expresses HLA class I antigen among HLA (Human Lymphocyteantigen) on their surface, but do not express HLA class II antigen. Whencell or tissue which has HLA class II antigen is allotransplanted,cellular immunity is activated by antigen presentation by HLA class IIantigen of donor cell or tissue and tranplanted cell or tissue isrejected. Therefore, when Muse cells which do not express HLA class IIantigen is allotransplanted, the rejection is difficult to occur andMuse cells can be engrafted. Thus, it is not required to use immunesuppressant when transplantation is carried out.

Further, Muse cells have immuno suppressive effect. That is, Muse cellscan suppress the induction of differentiation of monocyte tomonocyte-derived dendritic cell (MoDC) progenitor cell and the inductionof differentiation of monocyte-derived dendritic cell (MoDC) progenitorcell to dendrtic cell. Further Muse cells can suppress the activation ofT cells. These facts show that when Muse cells are allotransplanted,Muse cells suppresses immune reaction of a donor and are not rejected byimmune system of the donor. Accordingly, Muse cells can be engrafted anddifferentiate to many tissues and organs.

Muse cells can be used for cell therapy for allotransplantation. Musecells are usuful for regeneration medicine as described above.

Hereafter, the present invention is described in greater detail withreference to the following examples, although the present invention isnot limited to these examples

EXAMPLE 1 Preparation and Characterization of Muse-Enriched CellFractions and M-Clusters Materials and Methods

The following cells are used in Examples.

Two strains of human dermal fibroblast fractions (H-fibroblasts) andfour strains of human MSC (bone marrow stromal cell) fractions (H-MSCfractions) were used as mesenchymal cells. Human fibroblast fractionswere (1) H-fibroblast-1 (normal human fibroblast cells (NHDF), Lonza),and (2) H-fibroblast-2 (adult human dermal fibroblasts (HDFA, ScienCell,Carlsbad, Calif.)). Human MSC fractions, H-MSC-1, -2 and -3 wereobtained from Lonza, and H-MSC-4 was obtained from ALLCELLS. Human MSCfractions are specifically described in Pittenger, M. F. et al. Science284, 143-147 (1999); Dezawa, M. et al. J Clin Invest 113, 1701-1710(2004); and Dezawa, M. et al. Science 309, 314-317 (2005).

Cells were cultured at 37° C. in α-MEM (alpha-minimum essential medium)containing 10% FBS and 0.1 mg/ml kanamycin with 5% CO₂. Cells cultureddirectly after their shipment were considered to be the 1^(st) culture.When cells reached 95% confluence, cells were expanded at a ratio of 1:2(cell culture solution: medium). In this study cells from the 4^(th) to10^(th) subcultures were used.

Human ES cells (hESC) used herein were kyoto hESC-1 (KhES-1) obtainedfrom Kyoto University.

Mouse ES cells (TT2 cells) and human ES cells (KhES-1) were maintainedon mouse embryonic feeder (MEF) cells established from 12.5-day embryosof C57BL/6 mice.

Experiments were conducted by the following methods.

1. Stress Conditions for Mesenchymal Cells

To perform exposure to stress conditions including culture under poornutrition, culture under low serum, culture under low O₂,repetitive-trypsin incubations and long-term trypsin incubation, thefollowing six conditions were employed:

-   1) culture in non-serum containing medium (STEMPRO MSC SFM,    Invitrogen) for 2 days (serum free);-   2) culture in Hanks' Balanced Salt Solution (HBSS) buffer    (Invitrogen) for 2 days (HBSS);-   3) culture in 10% FBS in α-MEM combined with low O₂ (1% O₂) for 2    days (10%FBS+Low O₂);-   4) three consecutive 1-hr incubations (a total of 3 hours of trypsin    incubation) (Try 3×1 hr) in trypsin (0.25% trypsin-HBSS);-   5) long-term trypsin-incubation (LTT) for 8 hrs (LTT 8 hr); and-   6) LTT for 16 hrs (LTT 16 hr).

For negative controls, human peripheral mononuclear cell fractions wereused.

For conditions 4), 5) and 6), approximately 1×10⁵ to 5×10⁵ cells weresuspended in 5 ml trypsin solution, and incubated. Cells from stressconditions 1) through 3) were collected by a 5-min trypsin incubation,and cells from stress conditions 4) to 6) were transferred directly totubes.

Large numbers of dead cells resulting from stress conditions weredisrupted by vortexing. Specifically, 5 ml medium containing a maximumof 500,000 cells was transferred into a 15-ml Falcon tube, followed by 3min of vortexing at 1800-2200 rpm/min using a vortex mixer (IKA Works,Inc.). Centrifugation was performed at 2000 rpm for 15 min, so as toremove the supernatant. Collection efficiency of live cells aftervortexing ranged from approximately 70% to 80%.

2. MC Culture

In the Examples, cells were subjected to suspension culture inmethylcellulose-containing medium. Culture in methylcellulose-containingmedium is referred to as “MC culture.” MC culture is as described inNakahata, T. et al., Blood 60, 352-361 (1982).

Culture dishes were first coated with poly-HEMA (poly(2-hydroxyethylmethacrylate)) to avoid attachment of cells to the bottom of the dish.In brief, 600 mg of poly-HEMA (SIGMA) was dissolved in 40 ml of 95% EtOHby stirring at 37° C., added to the dish (e.g., 40 μl/well for 96-wellculture dish and 200 μl/well for 12-well culture dish), and the dish wasair-dried overnight.

MC (MethoCult H4100, StemCell Technologies) was suspended in 20%FBS+α-MEM to a final concentration of 2%. The cell concentration in thesemisolid MC medium was adjusted to be 8×10³ cells/ml at thisconcentration, so that the cell-to-cell distance was sufficiently largeto minimize cell aggregation. Cells and MC medium were mixed thoroughlyby gentle pipetting, and the mixture was transferred to apolyHEMA-coated dish. To prevent drying, a volume equal to one tenth ofthe initial MC culture of 10% FBS in α-MEM was gently added to the dishevery 3 days.

Cell clusters (referred to as Muse cell-derived embryoid body-like cellcluster=M-clusters since cell clusters were clusters from thepluripotent stem cells, Muse cells, of the present invention) werecloned on day 7. 0.01 M PBS was added to the medium, the cellscentrifuged at 2000 rpm for 20 min, and the supernatant discarded. Thisprocedure was repeated three times to wash the cells. The collected cellpellet was finally suspended in 10 μl of 0.01 M PBS containing TrypanBlue, applied to a glass slide, and the entire area was automaticallyimaged using phase contrast microscopy. Only multicellular clusterslarger than 25 μm that were negative for Trypan Blue and had anappearance similar to hES cells were counted as M-clusters. Thefrequency of M-cluster formation was calculated as the number ofM-clusters divided by the number of all live cells (all the TrypanBlue-negative cells). Since determination of the precise number of cellsin each M-cluster was difficult, each aggregate was counted as one cell,irrespective of its size.

For making cell clusters from hES cells, they were carefully isolatedfrom feeder cells so as not to include feeder cells, transferred to MCculture as described above, and imaged by phase contrast microscopy onday 3 of culture.

3. Single-Cell Suspension Culture

A 96-well dish was coated with polyHEMA as described above. Following alimiting dilution of cells with 10% FBS in α-MEM, single cells wereplated into each well. After plating, the actual number of cellsdeposited in each well was determined by visual inspection using a phasecontrast microscope. Empty wells or wells with more than one cell weremarked and excluded from the analysis. The calculation of M-clusterformation was performed on day 10 of culture. The frequency of M-clusterformation was calculated from 3 experiments for each strain with aminimum of 250 wells per experiment.

4. Alkaline Phosphatase Staining

M-clusters from H-fibroblast and H-MSC were washed several times with asufficient volume of saline. Staining was performed using the LeukocyteAlkaline Phosphatase kit (Sigma).

5. In vitro Differentiation of M-Cluster

After 7 to 10 days of MC culture or single-cell suspension culture,single M-clusters from H-fibroblast fractions and H-MSC fractions werepicked up with a glass micropipette and transferred onto agelatin-coated culture dish or cover glass. After another 7 days ofincubation, cells were dispersed from cell clusters. Cells weresubjected to immunohistochemical and RT-PCR analyses to determine thepresence or the absence of differentiation of the cells.

6. Immunohistochemistry

Cells were fixed with 4% paraformaldehyde in 0.01 M PBS. Muse-enrichedcell fractions and M-clusters both from H-fibroblasts and H-MSCfractions were collected by centrifugation, embedded in OCT compound,and 8 μm thick cryo-sections were cut. Cell clusters were fixed ongelatin-coated cover glasses and then subjected to immunohistochemicalanalysis.

The following primary antibodies against: Nanog (1:500, Chemicon),Oct3/4 (1:800, kindly provided by Dr. H. Hamada, Osaka University,Japan), Sox2 (1:1000, Abcam), PAR4 (1:100, Santa Cruz), SSEA-3 (1:20,DSHB), smooth muscle actin (1:100, Lab Vision), neurofilament M (1:200,Chemicon), α-fetoprotein (1:100, DAKO), mouse Numb-like (1:500, kindlyprovided by Dr. Yuh-Nung Jan, University of California San Francisco),type 1 collagen (1:20, Southern Biotech), Musashi, Nestin, Neuro D,MAP2, and Osteocalcin were used. Alexa 488-or Alexa 568-conjugatedanti-rabbit IgG, anti-mouse IgG or anti-mouse IgM antibodies (MolecularProbes, Carlsbad, CA) were used as secondary antibodies forimmunohistochemical analysis.

7. Determination of Karyotypes

Karyotypes of cells clonally expanded from M-clusters (obtained throughrepetition (1 to 3 times) of a cycle of collecting single cells fromM-clusters and then causing cells to form cell clusters again) both fromH-fibroblast fractions and H-MSC fractions were determined byquinacrine-Hoechst staining.

8. Injection of Cells into the Testes of Immunodeficient Mice

Naive cell fractions and Muse-enriched cell fractions and M-clustersboth from H-fibroblast fractions and H-MSC fractions were used.Muse-enriched cell fractions were prepared by adding serum to the cellsafter LTT and followed by three washes with 0.01 M PBS. M-clusters werecollected from MC cultures and also washed three times with PBS. 1×10⁵cells were suspended in PBS and injected using glass microtubes into thetestes of NOG mice (Registered trademark, mouse NOD/Shi-scid, IL-2RγKOJic, 8 weeks old, International Council for Laboratory Animal Science(ICLAS) Monitoring Center Japan). The average volume of the cells inM-clusters was measured using the 3D-graphic analysis software providedwith the laser confocal microscope (50 M-clusters were measured and thetotal volumes of the clusters were divided by the number of nuclei),which resulted in L5×10⁵ cells per 1 μl volume of collected M-clusterpellet. Each testis of a NOG mouse was then injected with the volumecorresponding to 1×10⁵ cells, and the mice were subjected to theexperiment for analysis 6 months after the injection.

As controls, 1×10⁶ mouse ES cells (for positive control, n=3) andmitomycin C-treated MEF cells (mouse embryonic feeder cells for negativecontrol, n=3) were injected into SCID mice testes, and the mice weresubjected to the experiment 8 weeks after the injection.

9. High-Resolution Analysis of Cells by Optical Microscope

Muse cells and M-clusters from H-fibroblast fractions and H-MSCfractions were observed using a stable high-resolution opticalmicroscope for cell types such as human MSC, fibroblasts, and neuronalcells. 10. Ultrathin sectioning for electron microscopy

M-clusters, SSEA-3(+) and SSEA-3(−) cells both from H-fibroblastfractions and H-MSC fractions as well as cell clusters formed by hEScells were centrifuged. The pellets were fixed with 2.5% glutaraldehydein 100 mM phosphate buffer (pH 7.2) for 30 min. The fixed samples wereembedded in 1% agar. The embedded samples were trimmed to 1 mm³, washedwith PBS, and stained with 2% OsO₄ in 100 mM phosphate buffer (pH 7.2)for 10 minutes at 4° C. The samples were washed with distilled water andthen stained with 5 drops of 2% uranyl acetate (UA) for 20 min at 4° C.After washing with distilled water, the stained samples wereincrementally dehydrated with 50%, 70% and 90% ethanol for 10 min eachat 4° C., and then completely dehydrated with three exchanges of 100%ethanol. The samples were incubated with propylene oxide for 5 min (forexchange) and embedded in 50% epoxy resin in propylene oxide for 60 min,followed by embedding in 100% epoxy resin and hardening at 60° C.overnight. Ultrathin sections were cut with a thickness of 70-80 nm andobserved in a 100 kV electron microscope using a CCD camera.

11. Growth Rate of M-Clusters

To calculate the fraction doubling time for cells in the M-clusters fromboth H-fibroblast fractions and H-MSC fractions, the clusters were eachtransferred to 96-well plates and treated with trypsin for 15 minfollowed by pipetting with a glass micropipette. The number of cells ineach well was counted. At least 20-30 M-clusters were analyzed atpredetermined time points (day 1, 3, 5, 7, 9, 10, 11, 12, 13 and 14).

12. RT-PCR

Naive cell fractions (about 10,000 cells per well of a 24-well plate)and cells in vitro differentiated from single M-clusters (1 to 3 cycles)both from H-fibroblast fractions and H-MSC fractions (about 10,000 cellsper well of a 24-well plate) were used. Total RNA was extracted andpurified using NucleoSpin RNA XS (Macherey-Nagel). First-strand cDNA wasgenerated using the SuperScript VILO cDNA Synthesis Kit (Invitrogen).The PCR reactions were performed using appropriate primers designed andEx Taq DNA polymerase (TaKaRa Bio Inc.). The used primers were asfollows.

Total RNA was extracted and purified using NucleoSpin RNA XS(Macherey-Nagel). First-strand cDNA was generated using the SuperScriptVILO cDNA Synthesis Kit (Invitrogen). The PCR reactions were performedusing appropriate primers designed and Ex Taq DNA polymerase (TaKaRa BioInc.). As positive controls, human fetal liver (Clonetech) was used forα-fetoprotein primers and human complete embryos were used for others.

13. Quantitative-PCR (Q-PCR)

Total RNA was collected from naive cell fractions, Muse-enriched cellfractions and M-clusters from H-fibroblast-1, H-fibroblast-2, H-MSC-1,and H-MSC-2 using the RNeasy Mini Kit (Qiagen GmbH) and cDNA wassynthesized using the RT² First Strand Kit (SA Biosciences). Customizedprimers were purchased from SA Biosciences and the DNA was amplified byquantitative PCR with the 7300 real-time PCR system (AppliedBiosystems). The data were processed using the ΔΔC_(T) method (Livak KJet al., Methods 25: 402-408, 2001).

14. DNA Microarray Analysis

Naive cell fractions, Muse-enriched cell fractions and M-clusters fromH-fibroblast-1, H-fibroblast-2, H-MSC-1, and H-MSC-2, as well as themixture of human peripheral mononuclear cells obtained from 4 healthyvolunteers were used. Total RNA was collected using the RNeasy Mini Kit(Qiagen) and analyzed by DNA microarray (TaKaRa Bio Inc.). Array signalswere processed and normalized using the Affymetrix Expression ConsoleV1.1 software. Pathway Studio 6.0 (Ariadne Genomics) was used to assignthe differentially expressed genes to functional categories in the GeneOntology. Hierarchical clustering was performed at a Euclidean distancebased on differentially expressed genes with average linkage clusteringby MeV4 (Saeed AI et al., Biotechniques 34(2): 374-378, 2003).

15. Detection of Telomerase Activity

Muse-enriched cell fractions and M-clusters from H-fibroblast fractionsand H-MSC fractions, and Hela cells were used. Telomerase activity wasdetected using the TRAPEZE XL telomerase detection kit (Millipore) andEx Taq polymerase (TaKaRa Bio Inc.). Fluorescence intensity was measuredwith a micro plate reader (Tecan).

16. Bisulfite Sequencing

Genomic DNA (1 μg) from naive cell fractions, Muse-enriched cellfractions, or M-clusters from H-fibroblast fractions and H-MSC fractionswas treated using CpGenome DNA modification kit (Chemicon). DNA waspurified using a QlAquick column (Qiagen). The promoter regions of humanOct3/4 and Nanog genes were amplified by PCR and then the PCR productswere subcloned into pCR2.1-TOPO. Up to 10 clones of each sample wereverified by sequencing using M13 universal primers, so that themethylation in each promoter region was detected. Primers described inShimazaki T et al., EMBO J, 12:4489-4498, 1993 were used for PCRamplification.

17. M-Cluster Formation Directly from Human Bone Marrow Aspirates

Three human bone marrow aspirates from healthy donors were purchasedfrom ALLCELLS. Mononuclear cell fractions were collected using theLymphoprep Tube (Axis-Shield PoC AS) and subjected to MC culturedirectly (without trypsin incubation) or after 8 hr-LTT as describedabove. M-clusters were counted on day 7.

18. MACS Sorting

Mononuclear cell fractions from human bone marrow aspirates of threehealthy donors (ALLCELLS) were first reacted with microbeads-conjugatedanti-CD105 antibody and sorted using MS Columns (Miltenyi Biotech).CD105(+) cells were collected as Fraction 1 (mesenchymal cellpopulation). CD105(−) cells were incubated with a mixture of anti-CD34and anti-CD117 antibodies conjugated to microbeads and sorted again toobtain CD34(+)/CD117(+) cells (Fraction 2 corresponding to ahematopoietic stem cell population) and CD105(−)/CD34(−)/CD117(−) cells(Fraction 3, the rest of the cell populations) (FIG. 5). The thuscollected samples were subjected to 8 hr-LTT and then the formation ofM-clusters was determined.

19. Flow Cytometry and Cell Sorting

Cells were incubated with phycoerythrin-labeled antibodies againstCD11c, CD29, CD34, CD44, CD45, CD49f, CD54, CD71, CD90, CD105, CD166,CD271 or vWF (Becton Dickinson) or with anti-SSEA-3 antibodies(Millipore). In the case of labeling with the anti-SSEA-3 antibody,cells were further incubated with FITC-conjugated anti-rat IgMantibodies. Calcium and magnesium-free 0.02 M PBS supplemented with 2 mMEDTA and 0.5% bovine serum albumin was used as the FACS antibodydiluents. Data were acquired and analyzed using FACSCalibur (BectonDickinson) and the CellQuest software or using FACSAria and the DIAsoftware. For cell sorting, cells were incubated with anti-SSEA-3antibody in the FACS antibody diluents and sorted by FACSAria (BectonDickinson) using a low stream speed and in the 4-way purity sortingmode.

20. Statistical Analysis

Data are expressed with average±SEM. Data were compared via pairedcomparison according to the Bonferroni method using ANOVA.

Results A. Stress Conditions for H-Fibroblast Fractions and H-MSCFractions

Examples of the results of exposure of H-fibroblast fractions and H-MSCfractions to stress conditions are shown in Table 1(below).

After exposing the cells to stress conditions and vortexing, Trypan Bluestaining was used to count the number of live cells, from which thesurvival rate was calculated. The surviving cells were collected andgrown in MC culture for 7 days. Stress conditions 2) resulted in a largenumber of dead cells and low efficiency for collecting surviving cells.It was therefore not possible to accurately determine the number offormed M-clusters, and the number of M-clusters for stress conditions 2)is thus denoted as ND (not determined) in Table 1.

Among the 6 stress conditions tested, the 16 hr-trypsin incubation wasmost effective for H-fibroblast fractions and the 8 hr-trypsinincubation for H-MSC fractions. When this experiment was repeated usingtwo strains of H-fibroblast fractions and four strains of H-MSCfractions, the same trend was observed. M-clusters could not berecognized in the negative control using human peripheral mononuclearcells. Examples of typical observed values are shown in Table 1.

TABLE 1 Survival rates after exposure to stress conditions and M-clusterformation in MC culture in H-fibroblasts, H-MSCs and human peripheralmononuclear cells. Cell cluster Survival formation in MC Start cellafter culture (>25 μm) number stress (%) (% to survived cells)H-fibroblast-1 1 Non-serum 30,000 75 7 2 HBSS 2,000,000 6 ND 3 10% FBS +LowO2 30,000 99 8 4 Tryp 3 × 1 hr 2,000,000 0.3 6 5 LTT 8 hr 2,000,000 115 6 LTT 16 hr 500,000 5 20 H-MSC-1 1 Non-serum 30,000 44 5 2 HBSS2,000,000 2 ND 3 10% FBS + LowO2 300,000 99 8 4 Tryp 3 × 1 hr 380,0000.9 9 5 LTT 8 hr 380,000 10 21 6 LTT 16 hr 500,000 3 14 Human peripheralmononuclear cells 5 LTT 8 hr 300,000 2 0 6 LTT 16 hr 300,000 1 0 ND (notdetermined): M-clusters could not be calculated accurately, because thefinal fraction contained a large number of dead cells and efficiency forcollecting surviving cells was low.

Among the 6 stress conditions tested, the 16 hr-trypsin incubation(H-fibroblast fractions) and the 8 hr-trypsin incubation (H-MSCfractions) were the most effective for the formation of M-clusters. Aseries of procedures including 16 hr- or 8 hr-trypsin incubationfollowed by vortexing at 1800-2200 rpm/min for 3 min and centrifugationat 2000 rpm for 15 min was termed “Long-Term Trypsin incubation (LTT)”and used for the enrichment of Muse cells. Collection efficiency of livecells after vortexing ranged from approximately 70% to 80% (FIGS. 6A and6B).

B. Criteria for M-Clusters

In the Examples, criteria for M-clusters were established. The averagediameter of single cells in Muse-enriched cell fractions both fromH-fibroblast fractions and H-MSC fractions was 10-13 μm (FIG. 7A). Whenthese cells were transferred to MC culture, the cells started to divide.The size of the individual cells became smaller after cell division, andthe gradually forming multicellular clusters comprised cells of 8-10 μmin diameter (FIGS. 8E and 8F). The size and appearance of the cells weresimilar to those of human ES cells subjected to MC culture (FIGS. 7B and7C). On day 7, most of the multicellular clusters became larger than 25μm, having a diameter of 100-150 μm. The cell clusters had an appearancevery similar to cell clusters formed by ES cells. Cell clusters largerthan 25 μm were collected using Φ25 μm filters (FIG. 7C) and thenanalyzed (up to 100 M-clusters each from H-fibroblast fractions andH-MSC fractions) by immunocytochemistry. Most of M-clusters werepositive for the pluripotency markers Nanog, Oct3/4, Sox2, PAR4 andSSEA-3 and also positive for alkaline phosphatase staining (FIGS.7E-7G). Pluripotency markers could be detected or not detected in cellclusters smaller than 25 μm, but their localization was sometimesatypical and the appearance of the cells was more similar to that ofcells in Muse-enriched cell fractions.

Based on these findings, only multicellular clusters larger than 25 μmin diameter were counted as M-clusters.

C. Analysis of Cell Clusters Generated from Human Mesenchymal CellFractions

It is well known that dormant tissue stem cells are activated when thetissue is exposed to stress, burdens or damages. In the Examples, H-MSCfractions and H-fibroblast fractions were exposed to stress conditionsby various methods. Specifically, the stress conditions tested were:treatment with non-serum medium; treatment with Hanks' Balanced SaltSolution (HBSS); treatment with low O₂ concentration; and long-termtrypsin incubation (LTT) for a total of 3, 8, or 16 hours. Cells thathad survived the stress conditions were collected and then suspended inmethylcellulose (MC)-containing medium (referred to as MC culture),followed by 7 days of MC culture at a density of 8000 cells/mL (FIG.8D). Each condition gave rise to cell clusters with sizes up to 150 μmin diameter (FIGS. 8E and F). FIG. 8C shows MC culture of H-fibroblast-1fractions on day 0, FIG. 8D shows MC culture of the same on day 7.Formation of the highest number of cell clusters were observed inH-fibroblast fractions subjected to 16-hr LTT and in H-MSC fractionssubjected to 8-hr LTT. FIGS. 8E and F show cell clusters (M-clusters)formed from H-fibroblast-1 fractions. FIG. 8E shows MC culture on day 7and FIG. 8F shows suspension culture of single cells on day 10. Cellclusters were sorted using filters with different pore-sizes accordingto their size, so that immunocytochemical analysis was performed. Thecell clusters with a diameter larger than 25 μm contained cells positivefor the pluripotent stem cell markers Nanog, Oct3/4, SSEA-3, PAR-4 andSox2 (FIGS. 9A-9F) and positive for alkaline phosphatase staining (FIGS.10A-10C). Electron microscopy revealed that cell clusters generated fromH-fibroblast fractions and H-MSC fractions had the same characteristicsas clusters formed by ES cells. The cells showed a similarnucleus/cytoplasm ratio, fewer organelles, and presence one or two bignucleoli in the nucleus (FIGS. 11A-11C).

Cells capable of forming cell clusters positive for pluripotency markersand alkaline phosphatase staining by suspension culture were found fromthe H-MSC fractions and H-fibroblast fractions of a living body. Thepresent inventors named these cells “Muse cells” (multilineagedifferentiating stress enduring cells). Cell populations formed fromH-fibroblast fractions subjected to 16 hr-LTT and H-MSC fractionssubjected to 8 hr-LTT are referred to as “Muse-enriched cell fractions(Muse-enriched cell populations).” Single cells obtained from the cellpopulations were subjected to suspension culture. Formation of M-clusterwas observed in 9%-10% of Muse-enriched cell fractions. This indicatesthat the muse-enriched cell fractions contained about 9%-10% Muse cells.

The growth of Muse cells isolated was examined. Cells began to divideafter 1-2 days in MC culture and kept dividing at a rate ofapproximately 1.3 days/cell division until day 10 (FIG. 13). However,cell growth gradually slowed down by days 11-12 and seized by around day14, with cell clusters that reached a maximum size of 150 μm indiameter. When the formed M-clusters were directly dissociated intosingle cells by a 5-min trypsin incubation and returned to single-cellsuspension culture, the cells remained alive but divided very slowly(5-7 days/cell division) or sometimes not at all (FIG. 12 (1); circle 1in the figure). Thus, once their proliferation has been limited (or oncetheir growth rate has been lowered), Muse cells do not re-acceleratetheir proliferation as long as they are maintained in suspensionculture. However, transfer of single M-clusters to adherent culturereinitiated cell proliferation. After 5-7 days, when relativelysmall-scale cell populations, at a stage of 3,000 to 5,000 cells, weredissociated by 5 minutes of trypsin incubation and subjected to MCculture, 40% of the cells formed new cell clusters (FIG. 12 (2); circle2 in the figure). When the clonally expanded cell populations wereallowed to reach a scale of about 5-10×10⁴ cells and then subjected toLTT to produce Muse cells (2^(nd) cycle) again, nearly 10% of thesecells formed M-clusters (FIG. 12). This culture cycle was repeated fivetimes, consisting of LTT-suspension culture-adherent culture, so thatevery cell generation showed the similar behavior and frequency ofM-cluster formation. The 5^(th) generation M-clusters (at the 5^(th)cycle) were still positive for pluripotency markers and alkalinephosphatase staining.

To confirm that these phenomena were not due to abnormal cells that hadundergone mutation or the like, karyotypes of cells were determined. Thekaryotypes of most cells clonally expanded from M-clusters were normaland did not show detectable chromosomal abnormalities (FIG. 14). Theseresults demonstrate that the phenomena resulted from normal cells.

These results demonstrate the capacity of Muse cells for self-renewaland clonal expansion. Muse cells grow through the series of cycle, “Musecells—M-cluster—clonal expansion.” It might be possible to obtain largenumbers of Muse cells from mesenchymal cell fractions.

D. Differentiation of M-Cluster into the Three Germ Layers

To confirm differentiation ability, single M-clusters were transferredonto gelatin-coated dishes and analyzed for differentiation. On day 7,a-smooth muscle actin (mesodermal marker), desmin (mesodermal marker),neurofilament-M (ectodermal marker), α-fetoprotein (endodermal marker),or cytokeratin-7 (endodermal marker) were detected (FIGS. 15A-15C).RT-PCR confirmed that 1^(st) and 3^(rd) generation M-clusters (1^(st) to3^(rd) cycles) expressed α-fetoprotein and GATA6 (endodermal marker),microtubule-associated protein-2 (MAP-2) (ectodermal marker), and Nkx2.5(mesodermal marker) while no differentiation was observed in naiveH-fibroblasts or MSC fractions cultured on gelatin-coated dishes (FIG.16).

Muse-enriched cell fractions, M-clusters, or ES cells were injected intothe testes of immunodeficient mice, so as to confirm teratoma formation(FIG. 17). Histological examination of the testes revealed that within 8weeks all mice injected with ES cells developed teratomas. However,remaining transplanted human cells and differentiation into various cellspecies were detected in the testes of 10 out of 13 mice injected withMuse-enriched cell fractions and 10 out of 11 mice injected withM-clusters as shown in FIG. 17. Teratoma formation was never detecteduntil at least 6 months in groups to which Muse cell-enriched cellfractions or M-clusters had been transplanted.

These data suggest that H-fibroblast fraction-, and H-MSCfraction-derived Muse cells, and M-clusters are capable ofdifferentiating into the 3 germ layers both in vitro and in vivo.

E. Quantitative PCR

Expression of markers relating to pluripotency and differentiation stateis shown in FIG. 18. The expression levels of Nanog were not so high inMuse-enriched cell fractions and cell clusters, compared with naivecells. Some pluripotent stem cells did not express Nanog at high levels(Chou YF et al., Cell 135, 449-461 (2008)); Bui HT et al., Development.135(23):3935-3945 (2008)). Similar to Nanog, Oct-4 was expressed at alow level in reprogramming somatic cells compared with mouse ES cells,as determined by Q-PCR (Bui HT et al., Development. 135(23): 3935-3945(2008)). Therefore, the expression levels of Nanog and otherpluripotency markers are not so important for pluripotency.

F. Gene Expression in Muse-Enriched Cell Fractions and M-Clusters

It was found by using quantitative PCR that some markers forpluripotency and an undifferentiated cell state were up-regulated tovarious degrees in both Muse-enriched cell fractions and M-clusters.Muse-enriched cell fractions merely contained 9%-10% Muse cells asdescribed above. However, compared to naive cell fractions, the genesshowed tendency of higher-degree or moderate up-regulation inMuse-enriched cell fractions: Rexl (ZFP42), Sox2, KLF-4, c-Myc, DPPA2(developmental pluripotency associated 2), ERAS, GRB7 (growth factorreceptor-bound protein 7), SPAG9 (sperm associated antigen 9), and TDGF1(teratocarcinomα-derived growth factor 1). The genes showed tendency ofhigher degree or moderate up-regulation in M-clusters were: DAZL(azoospermiα-like), DDX4 (VASA), DPPA4 (developmental pluripotencyassociated 4), Stella, Hoxb 1, PRDM1, and SPRY2 (sprouty homolog 2)(FIG. 18) compared with naive cells.

Overall gene expression in H-fibroblast fraction- and H-MSCfraction-derived naive cell fractions, Muse-enriched cells fractions,and M-clusters was compared with that in a human peripheral mononuclearcell fraction as a control. As a result, fluctuations in expressionpatterns of some genes were observed in naive cell fractions,Muse-enriched cells fractions, and M-clusters (FIG. 18).

Muse-enriched cell fractions and M-clusters showed low telomeraseactivity, suggesting that telomerase activity is not strongly related tothe proliferation activity of Muse cells (FIG. 19).

G. DNA Microarray Analysis of Global Gene Expression

Pearson correlation analysis of 108 probes was performed for humanperipheral blood mononuclear cells (as negative control), naive cellfractions, and the Muse-enriched cell fractions and M-clusters fromH-fibroblast fractions and H-MSC fractions (FIG. 20).

Also, odorant receptors and chemokine receptors expressed were picked upby DNA microarray analysis.

H. Muse Cells Exist In Vivo

The experiments described so far were performed with stable culturedcells, which may have acquired characteristics that differ from cells insitu when they are obtained from an adult body and then cultured. Hence,a possibility that Muse cells or M-clusters are artifact products cannotbe denied. Therefore, we attempted to directly obtain M-clusters from ahuman body; that is, human bone marrow cells without culture. Themononuclear cell fractions were isolated from human bone marrowaspirates and either cultured directly on MC (naive hBM-MC) or subjectedto 8 hr-LTT prior to MC culture (8 hr-hBM-MC; the survival rate ofmononuclear cells after LTT was about 3.5%). After 7 days, the cultureswere tested for M-cluster formation. 8 hr-hBM-MC formed M-clusters at afrequency of about 0.3%, approximately 75 times higher than that ofnaive hBM-MC (about 0.004%) (FIG. 21A). The M-clusters were positive foralkaline phosphatase staining (FIG. 21B). RT-PCR of cells clonallyexpanded from single M-clusters both from naive hBM-MC and 8 hr-hBM-MCshowed expression of α-fetoprotein, GATA6, MAP-2 and Nkx2.5 (FIG. 22).These results prove that Muse cells exist in vivo in human bone marrow,that they can be enriched by 8 hr-LTT and that they can form M-clusters.It was also confirmed that, among many cell types in bone marrow, themajority of Muse cells belong to the CD105(+) mesenchymal cellfractions.

As described above, Naive hBM-MC formed M-clusters at an extremely lowfrequency of about 0.004% in mononuclear cell fractions directlyisolated from the human bone marrow aspirate. Since it is conceivablethat culturing cells changes the composition of the cell population,cells in stable culture may have a different propensity to formM-clusters compared with naive mononuclear cells isolated from the bonemarrow. To confirm this possibility, a human bone marrow aspirate wascultured to collect primary MSCs and then the cells were directlysubjected to MC culture. This protocol resulted in a much higherfrequency of M-cluster formation of about 0.2%. When these primary MSCswere further cultured, to the 2^(nd) and 5^(th) subculture, thefrequency of M-cluster formation increased by about 0.5% and about 1.0%,respectively, with respect to naive cell fractions. Consistent with thisfinding, about 1.2% of naive H-fibroblast fractions and H-MSC fractionsformed M-clusters. These results suggest that Muse cells have highstress tolerance and can endure in vitro culture environment such assubculture procedures. Stable subcultured cell fractions thus showed ahigher frequency of M-cluster formation than mononuclear cell fractionsisolated directly from the bone marrow aspirate.

1. MACS Sorting

Bone marrow contains many cell types of mononuclear cells includingMSCs, hematopoietic lineage cells, and endothelial cells. To determinewhich fraction contains the Muse cells, mononuclear cell fractions wereisolated from a human bone marrow aspirate and then directly subjectedto MACS sorting using antibodies against CD34, CD117 (markers forhematopoietic cells) and CD105 (marker for MSCs) (FIG. 5). The fractionswere then treated with 8 hr-LTT, and the cells were grown in MC culturefor 7 days. Almost no M-clusters were detected in the CD34+/CD117+fraction, but the CD34−/CD117−/CD105+ fraction contained 50 times morecell clusters than the CD34−/CD117−/CD105− fraction. This resultsuggests that the majority of Muse cells belong to the CD105(+)mesenchymal cell fraction.

The three fractions from mononuclear cells from bone marrow containedthe following percentages of total cells: Fraction 1 (CD105+fraction):1.8%; Fraction 2 (CD34+/CD117+ fraction): 8.5%; and Fraction 3(CD34−/CD117−/CD105− fraction): 89.7%. The frequencies of cell clusterformation in Fractions 1, 2, and 3 were 0.5%, 0% and 0.01%,respectively. The formation of cell clusters in Fraction 1 was thusabout 50 times higher than that in Fraction 3.

J. FACS Sorting

As an example, FACS sorting was performed using SSEA-3 as a marker.

For both H-fibroblasts and H-MSCs, SSEA-3(+) and SSEA-3(−) cells wereseparated by FACS sorting and subjected to single-cell suspensionculture. Approximately 50%-60% of the SSEA-3(+) cells generatedM-clusters, while no M-clusters were formed from SSEA-3(−) cells.

Collection of SSEA-3 (+) Cells from the Adult Human Skin

It was attempted to directly isolate Muse cells from adult human skinwithout incubation of cultured cells or formation of M-clusters.

Human skin from healthy donors (n=3) was obtained from BIOPREDICInternational. The epidermis and fat tissues were carefully removed toseparate the dermis, and the dermis was incubated withCollagenase/Dispase in α-MEM containing 10% FBS for 36 hrs at 37° C.Cells were collected by filtering digested dermis, and were subjected tocentrifugation at 1500 rpm for 20 min, washed with α-MEM and incubatedwith 0.25% trypsin-HBSS for 5 min. Cells were further washed with FACSbuffer, and incubated with SSEA-3 for collecting SSEA-3 (+) cells bycell sorting using FACS. From about 7 cm² of the skin tissue,1.3±0.3×10⁴ single cells could be finally collected. SSEA-3(+) cellsaccounted for 1.7±0.2% of these collected single cells.

21.0±1.7% of SSEA-3(+) cells formed M-clusters within 7 days ofsingle-cell suspension culture by limiting dilution. The M-clusters werepositive for ALP staining, and RT-PCR showed that cells expanded from asingle M-cluster on gelatin-coated dishes expressed MAP-2, Brachyury,Nkx2.5, GATA6, and α-fetoprotein. These findings suggest that adulthuman dermis contains cells with the same properties as those of Musecells as in the case of adult human bone marrow aspirates.

Human adult dermis contains several types of stem or progenitor cells,such as SKPs (skin-derived progenitor cells), NCSCs (neural crest stemcells), melanoblasts (MBs), perivascular cells (PCs), endothelialprogenitors (EPs) and adipose-derived stem cells (ADSCs). To rule out apossibility that Muse cells are identical to one of these known stemcells, Muse cells were analyzed for expression of Snai1(markers forSKPs), Slug (markers for SKPs), Sox10 (markers for NCSCs), CD271(markers for NCSCs), Tyrp1 (markers for MBs), Dct (markers for MBs),CD117 (markers for MBs), CD146 (markers for PCs and ADSCs), NG2 (markersfor PCs), CD34 (markers for EPs and ADSCs), and von Willebrand Factor(markers for EPs). None of these markers were detected in SSEA-3(+)cells by FACS or RT-PCR analysis (FIGS. 33 and FIGS. 34A-34F),suggesting that Muse cells differ from these stem or progenitor cellsknown to be present in adult human dermis.

Phagocytic activity of Muse cells was determined using ferriteparticles. Muse cells easily incorporated ferrite particles, suggestingthat Muse cells have high phagocytic activity (FIGS. 35A and 35B).

K. Characteristic Features of Muse Cells

FACS analysis revealed that naive H-fibroblast fractions and H-MSCfractions contained fractions positive for CD44, CD49f, CD54, CD90, andCD105 expressed in mesenchymal cells, but were negative for CD30c, CD34,CD45, CD71, CD166, CD271, and von Willebrand (vWF) factors. Both naiveH-fibroblasts and H-MSCs contained about 0.7%-1.9% SSEA-3 (+) fractions(negative for CD44 and CD54) (FIG. 23).

SSEA-3 is one of the known markers for pluripotency. In naiveH-fibroblast fractions and H-MCS fractions, the frequency of M-clusterformation (about 1.2%) and the frequency of Muse-enriched cell fractionformation (9%-10%) were similar to the relevant percentage of SSEA-3(+)cells (naive cell fraction: about 0.7%-0.9%; and Muse-enriched cellfraction: 7%-8.3%). Such percentage of SSEA-3(+) cells may indicate astate of Muse cells. Immunohistochemical analysis revealed that thepercentage (number) of SSEA-3(+) cells in naive H-fibroblast fractionsand H-MSC fractions was less than 1%. SSEA-3(+) cells were then sortedfrom Muse-enriched cell fractions derived from H-fibroblast fractionsand H-MSC fractions and then subjected to single-cell suspensionculture. As a result, 50%-60% of the SSEA-3(+) cells generatedM-clusters. This result is about 6-7 times higher than that of M-clusterformation in Muse cell-enriched cell fractions and about 60 times higherthan that of M-cluster formation in naive cell fractions. Meanwhile,M-cluster formation was not observed in SSEA-3 (−) cell fractions. Ofnote, in clonally expanded cells (3000 to 5000 cells) from a single cellcluster derived from a FACS-sorted SSEA-3(+) cell fractions, about 45%of the cells were SSEA-3(+) (FIGS. 24A and 24B). This finding suggestedthat asymmetric cell division is involved in M-cluster formation andthat this can also be said in clonal expansion of a single M-cluster.Actually Numblike (known to be involved in asymmetric cell division)exists in only one of the two daughter cells after cell division (FIG.25). These results suggest that asymmetric cell division is involved inthe growth of Muse cells.

Electron microscopy revealed the presence of nuclear deformities andvacuoles in the cytoplasm inSSEA-3 (-) cells from H-fibroblast fractionsand H-MSC fractions sorted after LTT, indicating cell damage. Electronmicroscopy did not reveal clearly recognizable morphological differencesbetweenSSEA-3 (−) and SSEA-3(+) cells (FIGS. 26A and 26B).

The importance ofSSEA-3 (+) cells was also demonstrated in thetransplantation experiments. WhenSSEA-3 (−) cell fractions weretransplanted, very small number of cells were positive for the tissuemarkers compared to SSEA-3 (+) cell fraction transplantation.

The majority of SSEA-3(+) cells in Muse-enriched cell fractionsexpressed both Oct3/4 and Sox2, which were detected in the cytoplasm(FIGS. 27A and 27C), and a very small number of cells expressed in thenucleus (FIG. 27B). This result indicates that SSEA-3 can be a goodmarker for Muse cells. In contrast, in cells in M-clusters, Oct3/4 andSox2 predominantly localized to the nucleus (FIGS. 9B and 9F). It ispossible that this difference in the intracellular localization of thetwo markers reflects a difference in the cell status.

The possibility remains that Muse cells are artificially induced by LTT.As described above, the majority of Muse cells exist in the bonemarrow's CD105(+) cell fraction. Furthermore, SSEA-3(+) cells alsoshowed Muse cell properties. We therefore attempted to directly obtainMuse cells from adult human bone marrow aspirates by isolating them asSSEA-3/CD105 double-positive cells. Double-positive cells, whichconstituted 0.025% to 0.05% of bone marrow-derived mononuclear cells,were directly subjected to single-cell suspension culture without LTT.After 7 days, 11.4%±1.2% of the cells (corresponding to 0.003% to 0.005%of the mononuclear cells) formed M-clusters, which were ALP(+). SingleM-clusters were then again expanded by adherent culture to 3000 cellsand subsequently subjected to single-cell suspension culture. Of thesecells, 33.5%±3.1% cells formed 2^(nd) generation M-clusters, and RT-PCRof the cells that expanded from a single M-cluster on gelatin-coateddishes indicated the expression of α-fetoprotein, GATA6, MAP-2, andNkx2.5, suggesting that cells with properties consistent with those ofMuse cells reside in adult human bone marrow.

As described above, non-stem cells were removed by exposure to stressconditions, so that stem cells could be enriched. Muse cells could beefficiently collected by LTT and the following sorting of SSEA-3 (+)cells. Muse cells expressed pluripotency markers, were positive foralkaline phosphatase staining, and formed M-clusters capable ofdifferentiating into ectodermal, mesodermal, and endodermal cells. Also,Muse cells have no characteristics relating to tumorigenic proliferationand no telomerase activity, as revealed by measurement of the growthrate. This suggests that Muse cells have a multi-layered safety systemthat prevents a burst in proliferation. Such non-tumorigenic property ofMuse cells was also confirmed by an experiment wherein Muse cells wereinjected into mouse testes. This property is convenient for maintainingthe balance of biofunctions. Absence of such property may destroy aliving body due to abnormal growth or dysplasia, resulting intumorigenesis or teratoma formation.

The pluripotency of Muse cells did not become obvious in an adherentculture system, but was observed in suspension culture.

In general, it is thought that Muse cells are in a dormant state, butare activated in response to signals related to an acute crisis or tocontinued stressful conditions such as a severe injury, starvation, orischemia. Upon activation, Muse cells may contribute to tissueregeneration, intercellular interactions, and thus tissue organization.

EXAMPLE 2 Characterization of Muse Cells Isolated Using SSEA-3

In Example 1, SSEA-3 (+) cells were isolated from H-fibroblast and H-MSCby FACS sorting (Example 1, J). Examination in Example 1 revealed thatSSEA-3 (+) cell fractions obtained by FACS had the properties ofpluripotent stem cells; that is, they were Muse cells. Furthermore, invitro differentiation ability and in vivo differentiation ability wereexamined using isolated SSEA-3 (+) cells and then Muse-derived iPS cellswere established.

1. Examination of In Vivo Differentiation Ability by Transplantation ofCells into Damaged Tissures

SSEA-3 (+) Muse cells labeled with GFP (green fluorescentprotein)-lentivirus were isolated and then transplanted via intravenousinjection into immunodeficient mice (NOG mice) with damaged spinal cord(Crush injury of spinal cord), damaged liver (intraperitoneal injectionof CCl₄, fulminant hepatitis model), or damaged gastrocnemius (muscle)(cardiotoxin injection). Human skin cell-derived Muse cells were labeledwith GFP (green fluorescent protein)-lentivirus (Hayase M et al., JCereb Blood Flow Metab. 29(8): 1409-20, 2009) and then it was confirmedusing GFP that M-clusters were derived from the labeled cells. Crushinjury of spinal cord was performed at the level of Th9 (Farooque M etal., Acta Neuropathol., 100; 13-22, 2000) for NOG mice. Cardiotoxin wasinjected into the gastrocnemius (muscle) of the NOG mice to inducemuscle degeneration. Carbon tetrachloride was administered to NOG miceby peritoneal injection to induce liver degeneration. 1×10⁵ Muse cellswere transplanted by intravenous injection 2 days after for the muscleand liver and 7 days after for the spinal cord. Six mice were used foreach condition. Intact mice that received intravenous injection ofGFP-labeled MEC fraction were used as controls. At 3 or 4 weeks aftertransplantation, mice were fixed with 4% paraformaldehyde and thensubjected to immunohistochemical analysis and confocal laser microscopicobservation.

After 4 weeks, in mice with spinal cord injury, it was found that: cellspositive for GFP and human Golgi complex expressed neurofilaments (FIGS.28A and 28B); and that in mice with liver damage, cells positive for GFPand human Golgi complex (in regenerated liver) expressed human albumin(FIG. 28C). RT-PCR further confirmed the expression of human albumin inMuse cell-transplanted NOG mice liver (FIG. 29). GFP(+)cells injectedinto the regenerating muscle and at 3 weeks expressed human dystrophin(FIG. 30). In contrast to these results, transplantation of SSEA-3(−)human dermal fibroblast fractions resulted in a significantly smallernumber of integrated cells and fewer cells that were positive for therespective tissue markers. These findings suggest that Muse cells havean ability to integrate into damaged tissues and also to differentiatein vivo into ectodermal-, endodermal-, and mesodermal-lineage cells.

2. Differentiation of Expanded Cells Derived from M-Cluster Generatedfrom Single Muse Cells

It was examined whether an induction system is effective for regulationof differentiation of Muse cells. Single SSEA-3(+)-Muse cell-derivedM-clusters were transferred individually to adherent culture forexpansion. Expanded cells derived from a single Muse cell werecollected, divided into four fractions, and then each subjected toneural, osteocytes, adipocytes and hepatocyte inductions (n=5).

For neural induction, cells at a density of 1.0×10⁵ cells/ml werecultured in NEUROBASA1 medium (Gibco) supplied with B-27 Supplement inpoly-HEMA coated dish and cultured for 7 days for sphere formation. Fordifferentiation, spheres were transferred onto poly-L-Lysin-coatedglass, and incubated in 2% FBS supplied with 25 ng/ml FGF and 25 ng/mlBDNF for 10 days.

For osteocyte induction, cells at a density of 4.2×10³ cells/cm² wereincubated with osteocyte induction medium of Human Mesenchymal Stem CellFunctional Identification Kit (R&D Systems, SC-006) for 14 days.

For adipocyte induction, cells at a density of 2.1×10⁴ cells/cm² wereincubated with adipocytes induction medium of the human mesenchymal stemcell functional identification kit (R&D Systems) and incubated for 14days.

For hepatocyte induction, cells at a density of 2.0×10⁴ cells/cm² wereincubated with DMEM (+10% FBS, 10×ITS (GIBCO) supplied with 10 nMdexamethasone and 100 ng/ml HGF, 50 ng/ml FGF4) on collagen-coated dishfor 14 days.

Neural induction generated spheres containing cells positive for neuralstem cell markers nestin, Musashi and NeuroD (FIGS. 31A-D), whichfurther differentiated into MAP-2-or GFAP-positive cells when culturedin differentiation medium (FIG. 31E; 89±5.7% positive either for MAP-2or GFAP). Osteocyte induction produced cells positive for osteocalcin(97±3.5%) and alkaline phosphatase (FIGS. 31F-G). Adipocytedifferentiation produced cells with lipid droplets that stained with oilred (90±4.9%) (FIGS. 31H-I). Hepatocyte induction generated cellspositive for human α-fetoprotein (FIG. 31J; 87±7.6%), and RT-PCRconfirmed the expression of human albumin and α-fetoprotein (FIG. 32).These results demonstrate that Muse cells can be regulated (viainduction) to differentiate into cells of three lineages with very highefficiency.

3. Establishment of Muse-Derived iPS Cells (Muse-iPSC)

iPS cells are prepared by introduction of Oct3/4 gene, Sox2 gene, Klf4gene, c-Myc gene, Nanog gene, and Lin28 gene, for example. Muse cellshave properties analogous to iPS cells in that they express pluripotencymarkers and can differentiate into ectodermal, mesodermal, andendodermal cells. It was examined if Muse cells could be good materialsfor iPS cells.

A method employed for this purpose is as follows.

Four factors (Nanog, Oct3/4, KLF4, and c-Myc) were introduced intoSSEA-3(+) cells and SSEA-3 (−) cells from H-fibroblast fractions usingretroviral vectors according to the description of Takahashi et al.,Cell, 131, 861-872 (2007) and then cultured. The method is specificallydescribed as follows.

Establishment of Plasmid

The open reading frames of human Oct3/4, Sox2, Klf4, and c-Myc wereinserted into the pMXs retroviral vectors (Cell Biolabs).

Infection with Retrovirus and Establishment of iPS Cells

PLAT-A cells were seeded at a density of 5×10⁶ cells per 100-mm dish andthen cultured overnight. On the next day, transfection was performedusing Fugene HD. At 24 hours after transfection, medium exchange wasperformed. Supernatants were collected after 3 days and then filteredthrough a 0.45-μm filter. Polybrene (4 μg/ml) was then added.H-fibroblast fraction-derived SSEA-3(+) and (−) cells seeded at adensity of 1×10⁵ cells per 60-mm dish were infected with a virussolution. 24 hours later, the medium was exchanged with new mediumcontaining no virus. Cells were removed using trypsin on day 4 afterviral infection and then seeded on MEF (feeder cells) at a density of3×10⁴ cells. On the next day, the medium was exchanged with Primate ESmedium supplemented with 4 ng/ml bFGF. After 2 days, medium exchange wasperformed once every other day. After 30 days, colonies were picked upand then seeded over a 24-well plate.

PCR Analysis

RNA was purified using an RNeasy mini kit (QIAGEN). RNA (500 ng) wasreverse transcribed using SuperScriptll. Endogenous Oct, Sox2, Klf4,Myc, and Nanog primers, PCR conditions, and the like are as described inTakahashi et al., Cell, 131, 861-872 (2007).

In vitro iPS Cell Differentiation

iPS cells were collected using collagenase. Cell clusters were placed ondishes coated with Poly-HEMA and then cultured in DMEM/F12 mediumcontaining 20% Knockout serum replacement (Invitrogen), 2 mML-Glutamine, 1×10⁻⁴M nonessential amino acid, 1×10⁻⁴M 2-mercaptoethanol(Nacalai), and 0.5% Penicillin/Streptomycin. Medium was exchanged onceevery other day. 7 days later, embryoid bodies were seeded ongelatin-coated dishes, followed by 1 week of culture in the same medium.

Formation of Teratomas

iPS cells in a 60-mm dish were treated with a Rock inhibitor, collectedusing Accutase (registered trademark) in a tube, subjected tocentrifugation, and then suspended in PBS. These cells were injectedinto the testis of an NOG mouse (registered trademark) (CentralInstitute for Experimental Animals). After 12 weeks, the resultants werefixed with 4% paraformaldehyde. Paraffin sections were subjected to HE(Hematoxylin & Eosin) staining.

The following results were obtained.

Four factors, Nanog, Oct3/4, KLF4 and c-Myc were introduced intoH-fibroblast fraction-derived SSEA-3(+) and (−) cells using retroviralvectors according to the method described by Takahashi et al, Cell, 131,861-872 (2007). Cells were seeded again on MEF after 5 days, and thencultured. Just before colony pickup; that is, on day 30 of culture onMEF, all the generated colonies in SSEA-3 (−) cells were non-EScell-like colonies and none of them were positive for Tra-1-80 (ES cellmarker as well as reliable iPS cell marker). In contrast, many SSEA-3(+)cells formed colonies about eightfold the number of colonies formed bySSEA-3(−) cell fractions, which were positive for Tra-1-80. Importantly,genes tightly related to pluripotency, such as Nanog and Sox2 were allnegative inSSEA-3 (−) cells (all colonies and cells not forming colonieswere collected) even immediately before colony pickup on day 30 on MEF,as determined by RT-PCR. Meanwhile, SSEA-3(+) cells showed up-regulationof endogenous Oct3/4, KLF4, and Rexl and expressed Nanog and Sox2. Asexpected, SSEA-3(+) cells were subjected to colony pickup and thentransferred onto new MEF (feeder cells), iPS cells could be successfullygenerated at efficiency about 30 times higher than that of naiveH-fibroblast fraction cells. These iPS cells exhibited up-regulation ornew appearance of Tra-1-60, Tra-a-80, Rexl, UTF-1, telomerase reversetranscriptase (TERT) and factors expressed in human ES cells, asrevealed by immunocytochemistry, RT-PCR, and Q-PCR (FIGS. 36A-36F andFIG. 38). Furthermore, Nanog, Oct3/4, Sox 2, and TRA-1-81 were expressedin the thus obtained Muse cell-derived iPS cells (FIGS. 37A-37D). RT-PCRrevealed that Nanog, Oct3/4, and Sox 2 were expressed in Musecell-derived iPS cells, but not expressed in SSEA-3 (−) cell-derivedcolonies (FIG. 38).

The efficiencies for transduction of Oct3/4 gene, Sox 2 gene, Klf4 gene,and c-Myc gene were almost identical for the SSEA-3(+) and SSEA-3(−)cell fractions. Transduced cells with above four factors were thentransferred onto and cultured on mouse feeder cells at a density of1×10⁵ cells per dish. The generation of colonies were observed in bothfractions, but SSEA-3(+) cell fractions formed seven times more coloniesthan theSSEA-3 (−) cells. Furthermore, in contrast to those derived fromthe SSEA-3(+) cell fractions, none of the colonies derived fromtheSSEA-3 (−) cell fractions were found to be positive for the humanpluripotent stem cell marker TRA-1-81 even on day 30 of cultureimmediately before colony pickup (FIGS. 39A-39D). RT-PCR revealed thatendogenous Sox2 and Nanog were only expressed in SSEA-3(+) cell-derivedfractions but not in SSEA-3(−) cell fractions (FIG. 40).

All colonies generated from SSEA-3(+) and SSEA-3 (−) cell fractions werepicked and passaged in individual wells to establish iPS cell lines.After 3 passages, all colonies exhibiting human ES cell-like morphology(flat colonies) were individually subjected to RT-PCR (FIGS. 41A and B).Colonies expressing all three factors (endogenous Oct3/4, endogenousSox2 and Nanog) were counted iPS colonies. This analysis revealed thatonly colonies originating from SSEA-3(+)cells generated iPS cells andthe efficiency was 0.03%, while none of the colonies originatingfromSSEA-3 (−) cells generated iPS cells (FIGS. 41C and D).

Furthermore, iPS cells established from Muse cells differentiated intoectodermal, mesodermal, and endodermal cells, and formed teratomas inmice testes (FIGS. 42A and 42B, FIG. 43 and FIGS. 44A-44F).

The proliferation activity of Muse cells was not so high in terms ofgrowth rate and telomerase activity. Consistently, while Muse cellsdifferentiated into triploblastic cells in the mice testes, they did notdevelop into teratomas. This may be reasonable because if Muse cells aremaintained in adult human tissue such as in skin and bone marrow, theirproliferation should be strictly regulated, otherwise they would easilydevelop into tumors in virtually every part of the body. Moreover, evenpluripotent cells do not always show teratoma formation since epiblaststem cells cultured under certain conditions were demonstrated not toform teratomas in mice testes (Chou et al., Cell, 135, 449-461(2008)).As Muse cells originally showed some of the characteristics ofpluripotent cells such as pluripotency marker expression and theirdifferentiation ability, it is suggested that Muse cells could easilybecome iPS cells solely by an elevation of proliferative activity andformed teratoma in the mice testes. The induction mechanism of iPS isnot yet clarified, but procurement of tumorigenic proliferation in Musecells among mesenchymal cell fraction might be one of the possibilities.

iPS cells could be established at efficiency of about 0.001% from naivehuman dermal fibroblast fractions. This agrees with the report of K.Takahashi et al., Cell 131, 861 (2007). Therefore, iPS cell preparationefficiency from SSEA-3 (+) cells was 30 times higher than that fromnaive fibroblasts. This suggests that Muse cells mainly contribute toiPS cell generation.

Immunohistochemical analysis and RT-PCR analysis of embryoid bodies thatdeveloped from Muse-derived iPS cells showed that cells differentiatedinto ectodermal cells expressing neurofilament and MAP-2, mesodermalcells expressing SMA, Brachyury and Nkx2.5, and endodermal cellsexpressing a-fetoprotein and GATA-6 in vitro. Furthermore, injection ofMuse-derived iPS cells into testes of immunodeficient mice resulted interatoma formation. In contrast, testes injected with M-clusters did notdevelop teratomas for up to 6 months, and most were not significantlylarger than control testes that were injected with inactivated MEFs.

M-clusters and Muse-derived iPS cells were subjected to quantitative-PCR(Q-PCR). The results are shown in FIGS. 46 and 47. The expressionpatterns of genes related to cell cycle regulation differedsubstantially. Genes related to cell cycle progression were mostlydown-regulated in M-clusters but up-regulated in Muse-derived iPS cells.Expression of genes related to pluripotency and an undifferentiated cellstate were similar in M-clusters and Muse-derived iPS cells, but theexpression levels of Nanog, Oct3/4 and Sox2 were much lower inM-clusters than in Muse-derived iPS cells. Furthermore, cytosine guaninedinucleotides (CpGs) in the promoter regions of Nanog and Oct3/4 geneswere less methylated in Muse-derived iPS cells than in M-clusters, andthe promoter region of Nanog gene showed a lower CpG methylation levelin M-clusters than in naiveSSEA-3 (−) cell fractions (FIG. 45). Thisresult may partly explain the differences in the expression level ofpluripotency markers between Muse cells and Muse-derived iPS cells.

EXAMPLE 3 Pluripotent Stem Cell Derived from Fat Tissue of a Living BodyMaterials and Methods 1. Cell Materials

Two cell materials were used. One is cell which is commercial availableas a mesenchymal cell derived from human adipose tissue and the other isa mesenchymal cell established from the primary cultured cell of humansubcutaneous adipose tissue by the present inventors. The commercialavailable cell was the Human Adipose-Derived Stem Cell (HADSC) purchasedfrom Lonza (Lot.7F4309, 7F4089 and 7F4205). Cells were cultured inDulbecco's modified Eagle medium-High Glucose [DMEM, liquid(1×);Invitrogen Cat.11965-092] 15%(vol/vol) FBS [ES cell grade; HyClone] ,0.1 mg/mL Kanamycin sulfate [liquid(100×) ; lnvitrogen Cat.15160-054] at37° C.

The mesenchymal cell derived from human subcutaneous adipose tissue (inthe following text, they are called “established cells”) was establishedusing adipose tissue supplied by Tohoku University hospital, Japan usinga partially modified method of Estes BT et al. et al. (Nat Protoc.2010Jul:5(7):1294-1311).

Firstly, subcutaneous adipose tissue obtained aseptically was cut out topieces. Enzyme solution was prepared by adding lmg/mL colagenase Type I[100mg ; Worthington Biochemical Cat.LS004194], 1% (wt/vol) BSA [nacalai Cat.15111-45] into Phosphate-Buffered Saline [PBS, magnesiachloride and calcium chloride free]. The cut adipose tissue and theenzyme solution were mixed in equal amount in an centrifuging tube andextracellular matrix was digested by reacting the mixture for two hoursat 37° C. Two hours later, the mixture was centrifuged for 5 minutes at300 g and liquid phase between precipitate and supernatant was aspiratedand undigested extracellular matrix was removed from the precipitate andsupernatant using 100 μl cell strainer. Then, DMEM [15% (vol/vol) FBS,0.1 mg/mL kanamycin sulfate] was added and the solution was centrifugedfor 5 minutes at 300 g and the supernatant was removed. The precipitatewas suspended to be seeded. Twenty four hours later from theinoculation, culture medium was replaced.

For the commercially available cell and the established cell, culturemedium was replaced every two days. When the cultured reached to 90%confluent, the cells were sub-cultured at the ratio of 1:2. For mouse EScells, 15% (vol/vol) FBS [ES cell grade ; HyClone], 0.1 mg/mL kanamycinsulfate[ liquid(100×); Tnvitrogen Cat.15160-054], 0.1 mM MEMNon-Essential Amino Acids Solution [NEAA, liquid 10 mM(100×); InvitrogenCat.11140-050]

Sodium pyruvic acid solution 1 mM [SP, liquid 100 mM(100×); InvitrogenCat.11360-070], 1000 U/mL Leukemia Inhibitory Factor [LIF, ESGRO], 100μM 2-mercaptoethanol was used and MEF (mouse embryonic feeder) treatedwith mytomycin was used as feeder cells. MEF prepared from a 12.5-dayold C57BL/6 mouse fetus.

2. FACS

FACS analysis for the commercial available human mesenchymal cellderived from human adipose tissue and mesenchymal cell established fromthe primary cultured cell of human subcutaneous adipose tissue wascarried out. Cells were used for FACS analysis when the culture reachedto 100% confluent. Firstly, medium was removed from a dish and 3 mL oftrypsin-EDTA was added to the dish and expanded to all cells. Then, thecells were incubated for 10 minutes at 37° C. Confirming that the cellswere detacched from the dish with certainty, 1 mL of FBS was added andput cells into single cell by pipetting. Then, cells were recovered andcounted. Cells were suspended such that the cell density became onemillion cells per 100 μl. Cells were incubated with 10% inactivatedhuman serum for 20 minutes on ice and FcR blocking was carried out.Then, the cells were incubated with anti-SSEA-3 antibody (1:50,Millipore) and FITC-labeled anti-rat IgM antibody (1:100, JacksonImmunoresearch) used as a primary antibody and a secondary antibody,respectively, to be stained. After the staining, analysis and sortingwere carried out using Special Order Research Products FACS Arial I(Becton Dickinson). FIGS. 49A-49C show the results. Peaks of the sampleto which the secondary antibody (FIG. 49B) only was added and the samplewhich was SSEA-3 stained (FIG. 49C) were adjusted such that the positionof the peaks was the same with that of non-staining sample (FIG. 49A).Then, cell fraction showing almost the same FITC intensity asnon-staining sample was recovered as non-Muse cells (P6 area in FIGS.49A-49C) and cell fraction showing the stronger FITC intensity than thesample with the secondary antibody only was recovered as Muse cells (P3area in FIGS. 49A-49C).

3. Single Cell Suspension Culture

Cells which were sorted by FACS using the expression of SSEA-3 as amarker from the commercial available human mesenchymal cell derived fromhuman adipose tissue and mesenchymal cell established from the primarycultured cell of human subcutaneous adipose tissue were stained withtrypan blue and living cells were counted. Limiting dilution wasperformed to adjust to lcell/well and cells were suspended in a MEM with15% (vol/vol) FBS and seeded in 96 well dish coated with poly-HEMA.Suspension culture was carried out for 7 to 10 days and the formationrate of M-clusters was counted.

4. Differentiation of M-Cluster In Vitro

M-clusters which had been cultured for 7 to 10 days in the single cellsuspension culture of 3. above were transferred to 24 well dish coatedwith 0.1% gelatin into which 300 μl of α MEM [15% (vol/vol) FBS)] wasadded such that number of M-clusters was adjusted to one M-cluster/well.Several hours later, 200 μl of culture medium was added. After one ortwo week-culture, cells were analyzed by immunocytohistochemistry andRT-PCR.

5. Immunocytohistochemistry

M-cluster obtained in 3. above was fixed by 4% (vol/vol)paraformaldehyde/0.01M PBS and enbedded with O.C.T. compound to preparefrozen sections which was to be immunostained. M-cluster whichdifferentiated on a cover glass coated with gelatin was also fixed by 4%(vol/vol) paraformaldehyde/0.01M PBS and used for immunostaining. AntiSSEA-3 antibody [1:50, Millipore], anti Nanog antibody [1:100,Millipore], anti Oct3/4 antibody [1:100, Santa Cruz], anti Sox2 antibody[1:1000, Millipore], anti PAR4 antibody (1:100, Santa Cruz), anti smoothmuscle actin (SMA) antibody [1:100, Lab Vision , anti Neurofilament-Mantibody [1:100, Millipore] and anti Cytokeratin 7 antibody (CK7)(1:100, Milllipore] were used and these primary antibodies were detectedusing FITC-, Alexa-488 or Alexa-568 labeled anti-rabbit IgG, anti mouseIgG or anti-mouse IgN [1:500, Molecular Probes].

6. RT-PCR

Total RNA was extracted from M-cluster differentiated in vitro by themethod mentioned in 4. above using Nucleo Spin RNA Xs (Macherey-Nagel).cDNAs were obtained by reverse transcription using Super Script VILOcDNA Synthesis Kit (Invitrogen) to obtain cDNA from the total RNA. ForPCR, Ex taq DNA polymerase (Takara Bio, Inc.) was used to examine theexpression of α-fetoprotein (AFP, a marker for endoderm), GATA6 (amarker for endoderm), micro tubule-associated protein-2 (MAP-2) (amarker for ectoderm) and Nkx2.5 (a marker for mesoderm). FIG. 62 showsthe sequence of the used primers. Human whole embryo, human fetus liver,human adult brain and human adult liver were used for control.

7. Transplantation of Cells to Testis of Immunodeficient Mice

SSEA-3 positive cells prepared using FACS as mentioned in 2. above werestained with trypan blue and living cells were counted. After counted,cells were suspended to 1.5×10⁵ cells/μL and 1.5×10⁵ cells weretransplanted to one testis of 8-weeks old male CB17/Icr-Prkdcscid/CrlCrlj (SCID) mice. Mouse ES cells were transplanted as positivecontrol and PBS was administered for negative control. Mice in SSEA-3positive group were refluxed and fixed 4 months and 6 months after thetransplantation and mice in mouse ES cell transplantation group and PBSadministration group were refluxed and fixed 2 months after thetransplantation.

8. Study for the Self-Renewal Ability

First generation of M-cluster which was obtained by the single cellsuspension culture of SSEA-3 positive cells prepared using FACS asmentioned in 2. above was subjected to attached culture in a dish coatedwith 0.1% gelatin and containing 300 μl of α MEM [15% (vol/vol) FBS] togrow cells. Growing cells were detached by adding trypsin-EDTA and cellswere subjected to the single suspension culture again to form 2ndgeneration of M-cluster. FIG. 60 shows the summary of the method.

Results

In the present example, 2 sources, one is cell which is commerciallyavailable as human adipose derived mesenchymal cell and the other wasmesenchymal cell established by the present inventors from subcutaneousadipose tissue. Using the method of the present example, cells werecollected from human subcutaneous adipose tissue the volume of which was15.4cm³ (FIG. 51A) and were cultured for 16 days. As human adiposetissue derived mesenchymal cells (FIG. 51B), 1.96×10⁷ cells wereestablished.

FACS analysis revealed that Muse cells which can be separated usingSSEA-3 as a marker are present in each cell fraction source. Forcommercially available cells, 4.3±0.36% Muse cells are contained in thecell fraction and 7.3±0.37% Muse cells are contained in the cellsestablished from the primary culture of the adipose tissue (FIGS. 52Aand 52B). FIG. 52A shows the expression of SSEA-3 in commerciallyavailable human adipose-derived stem cell (HADSC) and FIG. 52B shows theexpression of SSEA-3 in the established cell.

Muse cell forms M-cluster (Muse cell-derived embryoid body (EB)-likecell cluster) which is similar to embryoid body from a human ES cellfrom a single cell by suspension culture. Thus, Muse cell positive forSSEA-3 and non-Muse cell negative for SSEA-3 were collected andsubjected to suspension culture. As a result, it was confirmed thatM-cluster was formed from single Muse cell only. From SSEA-3 positivecell isolated from commercially available HADSC, 65 to 66% cells wereformed M-cluster and 44.2% cells formed M-cluster from SSEA-3 positivecells isolated from established cell. FIGS. 53A and 53B show themorphology of M-cluster. FIG. 53A shows the morphology of M-clusterobtained from commercially available HADSC and FIG. 53B shows themorphology of established cell.

Further, the expression of microtubule-associated protein-2 (MAP-2) (amarker for ectoderm), GATA6 (a marker for endoderm), α-fetoprotein (AFP,a marker for endoderm), and Nkx2.5 (a marker for mesoderm) was confirmedfor in vitro culture of the formed M-cluster in a dish coated withgelatin by RT-PCR. The expression shows the spontaneous differentiationinto cells of three germ layers (tridermic cells) (FIG. 54). In FIG. 54,M-cluster I shows the differentiation of M-cluster obtained fromcommercially available HADSC and M-cluster shows the differentiation ofM-cluster obtained from the established cells.

Further, in immunostaining using antibodies against Neuro filament-M(NF, ectodermal nerve), Cytokeratin 7 (CK7, endodermal biliary tree) andSmooth muscle Actin (SMA, mesodermal smooth muscle), it was proved thatM-cluster differentiated into cells of three germ layers. In FIGS.55A-55C, the oval objects are nucleus stained by DAPI and elongatedobjects are cells which are stained with anti-NF antibody, anti-CK7antibody and anti-SMA antibody.

It was further confirmed that M-cluster expressed pluripotent markerssuch as Nanog, Oct3/4, PAR4, Sox2, SSEA-3 and alkaline phosphatase(ALP). These results proved that Muse cell isolated from HADSC haspluripotency and ability to differentiate into cells of three germlayers (FIG. 56). In FIG. 56, left side panels show nucleus whichstained blue, central panels shows immunostained images stained withantibodies against Nanog, Oct3/4, PAR4, Sox2 and SSEA-3 from on high,respectively. Right side panels are merged images of DAPI stained imagesand imunostained images. The lowermost panel shows images stained withanti ALP antibody, which indicates the expression of ALP.

For the transplantation of cells into mouse testis, mouse ES cellsformed teratoma in 8 weeks after the transplantation. While, Muse cellsdid not form teratoma for 4 months after the transplantation. This showsthat Muse cells derived from commercially available HADSC and theestablished cells were not tumorigenic (FIGS. 57A and 57B). In FIGS. 57Aand 57B, A shows the results of control (Intact) and the transplantationof Muse cells)(4 months), and B shows the results of the transplantationof mouse ES cells (8 weeks).

Further, it was confirmed by HE—stained image that mouse ES cells formedteratoma including tridermic cells (FIGS. 58A-58D). In FIGS. 58A-58D, I(FIG. 58B) shows intestinal epithelium (endoderm), II (FIG. 58C) showsneural elements (ectoderm) and III (FIG. 58D)shows smooth muscle(mesoderm). While, in testis to which Muse cells were transplanted,HE-stained image did not reveal the formation of tumor (FIGS. 59A and58B).

Self-renewal ability was examined by the method mentioned in 8. above.The formation rate of the 1st generation M-cluster was 35.5% and theformation rate of the 2nd generation M-cluster was 29.7% (FIG. 60).These results show that the obtained Muse cells had self-renewalability.

EXAMPLE 4 Pluripotent Stem Cell Derived from Umbilical Cord

Muse cells were isolated from human umbilical cord using SSEA-expressionas a marker by the same method as mentioned in 1. to 3. of Example 3.Human umbilical cord was collected with the consent of a patient.Umbilical cord was disinfected and blood vessels and epidermis on thesurface were removed and only inner mesenchymal tissue was collected.The collected tissue was cut into pieces. Mesenchymal cells wereobtained by subjecting the cut tissue to attached culture. The obtainedmesenchymal cells were subjected to FACS to collect Muse cells as SSEA-3positive cells. The obtained cells were subjected to single cellsuspension culture to form M-clusters and M-clusters were analyzed byRT-PCR and immunocytohistochemistry. FIGS. 61A and 61B show themorphology of mesenchymal cells obtained from human umbilical cord. FIG.62 shows the expression of SSEA-3 on mesenchymal cells obtained fromhuman umbilical cord. The mesenchaymal cells were obtained from humanumbilical cord by the same method as mentioned in 2. of Example 3. FIG.63 shows the morphology of M-cluster formed from SSEA-3(+) cell isolatedfrom human umbilical cord. FIG. 64 shows the expression of alpha fetoprotein (AFP, a marker for endoderm), GATA6 (a marker for endoderm),micro tubule-associated protein -2 (MAP-2, a marker for ectoderm) andNkx2.5 (a marker for mesoderm) in cells derived from a single SSEA-3(+)cell-derived cell cluster in gelatin culture.

Through examples 3 and 4, it was confirmed that Muse cells which arepluripotent cells are present in human adipose tissue derivedmesenchymal cell and human umbilical cord-derived mesenchymal cell.

Muse cells express pluripotent markers and have ability to differentiateinto tridermal cells from a single cell. Accordingly, human adiposetissue is prospective source of autologous cell transplantation therapy.Considering actual therapy, it is easier to collect adipose tissue froma recipient than to collect bone marrow or skin from the recipient. Inthe establish method of Muse cells of the present example, 1.9×10⁷ cellswere obtained as mesenchymal cells by 16-days culture of cells collectedfrom human adipose tissue the volume of which was 15.4cm³. Provided thatthe positive rate for SSEA-3 is 5%, 9.8×10⁵ cells among 1.9×10⁷ cellsfrom adipose tissue are estimated as Muse cells. Since sufficient numberof cells can be prepared in short time from lower amount of tissue, itis extremely preferable for autologous transplantation therapy. Musecells derived from human adipose tissue derived mesenchymal cells arecells from living body which is different from human ES cells and humaniPS cells Thus, it is expected that Muse cells would solve the problemssuch as affection by gene introduction and risk for tumor formationwhich are confronted by other pluripotent cells.

The same thing can be said for Muse cells isolated from mesenchyamalcells derived from umbilical cord.

EXAMPLE 5 Expression of HLA Class Antigens on SSEA Positive CellsDerived from Human Bone Marrow Stroma Cells and Human DermalFibroblasts; Analysis by Flowcytometry

The expression of HLA class I antigen and HLA-class II antigen on SSEA-3positive cells derived from human bone marrow stroma cells wereconfirmed by flowcytometry. FIG. 65 shows the results. As shown in FIG.65, HLA 1 was positive for human bone marrow stroma cells, but HLA IIwas negative.

The expression of HLA class I antigen and HLA-class II antigen on SSEA-3positive cells derived from dermal fibroblasts were also confirmed. FIG.66 shows the results. As shown in FIG. 66, HLA 1 was positive for humandermal fibroblasts, but HLA II was negative.

EXAMPLE 6 Expression of HLA Class Antigens on Muse Cells; Analysis byImmunocytochemistry

The expression of HLA class I antigen on Muse cells (SSEA-3 positive)and non Muse cells (SSEA-3 negative) was examined by immunocytochemistrystaining using anti-human HLA-ABC antibodies (eBioscience). Anti-mouseIgG antibody was used as a second antibody (labelled with Alexa568).FIG. 67 shows the results. As shown in FIG. 67, the expression of HLAclass I antigens was observed on both of Muse cells (SSEA-3(+) cells)and non-Muse cells (SSEA-3(−) cells).

The expression of HLA class II antigen on Muse cells (SSEA-3 positive)and non-Muse cells (SSEA-3 negative) was examined by immunocytochemistrystaining using anti-human HLA-DR antibody (eBioscience). Anti-mouse IgGantibody was used as a second antibody (labelled with Alexa568). FIG. 68shows the results. As shown in FIG. 67, the expression of HLA class IIantigens was not observed on both of Muse cells and non-Muse cells.

FIG. 69 shows the non-specific reaction in the above experiment. Asshown in FIG. 69, the light emission by Alexa 568 was not observed. Thisresult shows that there was no non-specific reaction of Alexa568.

EXAMPLE 7 Study for Immune Suppression Effect of Muse Cells byLymphocyte Stimulation Test

The present inventors confirmed whether or not Muse cells had immunesuppression effect by the tests for the induction of dendritic cells.More specifically, monocytes were isolated from human peripheral bloodand co-cultured with Muse cells. Through this study, the inventorsconfirmed whether or not dendritic cells were induced from monocytes.

Muse cells and non-Muse cells were isolated from human fibroblasts usingSSEA-3 Human monocytes were also used.

Regarding culture medium, α MEM (10% FBS, 2 mM .L-glutamine, Kanamycin)was used for Muse cells and non-Muse cells isolated from humanfibroblasts, RPMI-1640 (10% FBS) was used for induction of humanmonocytes into monocyte-derived dendritic cell (MoDC) progenitor celland RPMI-1640 (10% FBS, 2 mM L-glutamine, 2 mM sodium pyruvate, 40 ng/mLGM-CSF, 20 ng/mL IL-4, Kanamycin) was used for induction of MoDCprogenitor cell to dendritic cells.

Specific methods are as follows.

(1) Collection of Monocytes

Peripheral blood of healthy subject was collected and monocytes fractiononly was isolated using Lymphoprep Tubes (Axis-Shield PoC AS). Then, themonocytes fraction was suspended in RPMI medium (10% FBS) and seeded in10 cm dish. Next day, the dish was washed two times to remove unattachedcells.

Attached cells were detached from the dish using 0.25% trypsin/2 mM EDTAand stained with anti-human CD14 (a marker for human monocyte) to beanalyzed by FACS. FIG. 70 shows the results. It was confirmed that morethan about 90% cells were CD14 positive.

(2) Induction of Differentiation from Monocyte to MoDC Progenitor Cells

When monocytes are cultured in the presence of GM-C SF and IL-4 forseveral days, the cells differentiate into MoDC progenitor cells andthen becomes mature dendritic cells by TNF-α.

In the presence of Muse cells, in the presence of non-Muse cells,monocytes isolated as mentioned in (1) above were cultured. The mediumused for the induction of the monocyte-derived dendritic cell (MoDC)progenitor cells by culturing monocytes was RPMI-1640 (10% FBS, 2 mML-glutamine, 2 mM sodium pyruvate, 40 ng/mL GM-CSF, 20 ng/mL IL-4,Kanamycin). In the culture, conditions were changed for the absence orpresence of cytokines and the absence or presence of co-culture withMuse cells or non-Muse cells. The specific conditions were (A) onlymonocytes (without cytokines)(Negative control), (B) only monocytes(with cytokines) (Positive control), (C) monocytes+Muse cells (SSEA-3(+)cells), and (D) monocytes+non Muse cells (SSEA-3(−) cells). Theexpression “monocytes+Muse cells” means co-culture of monocytes withMuse cells. Five days later, cells were detached using 0.25% trypsin/2mM EDTA and stained with anti-CD1a (a marker for human MoDC progenitorcells) antibody and anti-CD14 antibody (a marker for human monocyte) tobe analyzed by FACS. FIGS. 71A-71E show the results. FIGS. 71A, B, C andD show the results of FACS analysis for conditions (A), (B), (C) and(D), respectively. In FIGS. 71A-71E, Q1 shows the presence of monocytes,Q2 shows the presence of halfway-differentiated cells, Q3 shows othercells, and Q4 shows MoDC progenitor cells (See FIG. 71 E). FIG. 72 showsthe ratio of CD14 positive cells (monocytes) to CD1a positive cells(MoDC progenitor cells) after culture for each condition. As FIG. 72shows, in case where monocytes were cultured in the absence of cytokines(condition (A)), MoDC progenitor cells were little. However, in casewhere monocytes were cultured in the presence of cytokines (condition(B)), MoDC progenitor cells were observed. When monocytes wereco-cultured with non-Muse cells, the number of MoDC progenitor cells wassmaller than condition (B). While, monocytes were co-cultured with Musecells, induction of MoDC progenitor cells was significantly suppressedcompared to condition (B). These results show that Muse cells suppressedinduction of differentiation of MoDC progenitor cells from monocytes.Both of Muse cells and non-Muse cells suppressed induction ofdifferentiation of MoDC progenitor cells. However, the induction by Musecells was stronger.

(3) Induction of Differentiation from MoDC Progenitor Cells to DendriticCell

The monocyte-derived dendritic cell (MoDC) progenitor cells were inducedby the culture of monocytes using RPMI-1640 (10% FBS, 2 mM L-glutamine,2 mM sodium pyruvate, 40 ng/mL GM-CSF, 20 ng/mL IL-4, Kanamycin) of (2)above. Five days after monocyte culture started, 10 ng.mL human TNF-αwas added to the culture medium. In the culture, conditions were changedfor the absence or presence of cytokines and the absence or presence ofco-culture with Muse cells or non-Muse cells. One μg/mL cyclosporine Ainstead of TNF-α was added to the culture medium as a control sincecycloporine A inhibits the maturation of dendritic cells. Further, sevendays after monocyte culture started (2 days after the TNF-α treatment),cells were stained with anti-human CD86, which expresses strongly ondendritic cell to be analyzed with FACS. FIGS. 73A-73G show the results.FIGS. 73A-73G ((i) to (vii)) shows the results under conditions (i) onlyMoDC progenitors (without cytokines), (ii) only MoDC progenitors (withcytokines), (iii) only MoDC progenitors (with cytokines+Cyclosporine A),(iv) MoDC progenitors+Muse cells (SSEA-3(+) cells), (v) MoDCprogenitors+Muse cells (SSEA-3(+) cells+Cyclosporine A, (vi) MoDCprogenitors+non-Muse cells (SSEA-3(−) cells) and (vii) MoDCprogenitors+non-Muse cells (SSEA-3(−) cells+Cyclosprorine A),respectively. As the figure shows, the expression of CD86 reduces whenMuse cells were co-cultured with MoDC progenitor cells. FIG. 74 showsthe expression of CD86 after the culture for each condition (ii) to(vii). Each condition is (ii) MoDC progenitors only, (iii) MoDCprogenitors+Cyclosporine A, (iv) MoDC progenitors+Muse cells (SSEA-3(+)cells), (v) MoDC progenitors+Muse cells (SSEA-3(+) cells)+CyclosporineA, (vi) MoDC progenitors+non-Muse cells (SSEA-3(-) cells) and (vii) MoDCprogenitors+non-Muse cells (SSEA-3(-) cells)+Cyclosporine A). As thefigure shows, the expression of CD86 reduces when Muse cells wereco-cultured with MoDC progenitor cells. These results show that Musecells suppressed induction of differentiation of monocytes tomonocyte-derived dendritic cell (MoDC) progenitor cells and MoDCprogenitor cells to dendritic cells. Both of Muse cells and non-Musecells suppressed induction of differentiation of monocytes and MoDCprogenitor cells, the extent of suppression was stronger for Muse cells.

INDUSTRIAL APPLICABILITY

According to the present invention, pluripotent stem cells can beobtained from body tissue without using any germ cells or early embryosand without using an artificial induction operation such as foreign genetransfer or introduction of a specific compound. The pluripotent stemcells of the present invention can be efficiently prepared without usingan artificial operation such as foreign gene transfer, so that they canbe safely used when applied for treatment. Also, the pluripotent stemcells of the present invention can be used for regeneration medicine andtreatment for dysfunctional tissue or the like, and they can be furtherused for research into cell division or tissue regeneration, forexample.

All publications, patents, and patent applications cited in thisspecification are herein incorporated by reference in their entirety.

1-3. (canceled)
 4. A cell fraction comprising at least 30% isolated SSEA-3(+) pluripotent cells (SSEA-3 (+) Muse cells) characterized by being positive for SSEA-3(+) and negative for at least one member selected from the group consisting of (a) CD 117 and CD 146; (b) CD117, CD146, NG2, CD34, vWF, and CD271; (c) CD34, CD117, CD146, CD271, NG2; and vWF, Sox10, Snai1, Slug, Tyrp1, and Oct; and which: (a) exhibit telomerase activity no greater than that of human fibroblast; (b) do not exhibit neoplastic growth; (c) are capable of differentiating into three germ layers in vivo and in vitro ; and (d) are capable of self-renewal.
 5. A cell fraction comprising at least 30% isolated SSEA-3(+) pluripotent cells (SSEA-3 (+) Muse cells) characterized by being positive for CD 105 and negative for at least member selected from the group consisting of (a) CD 117 and CD 146; (b) CD117, CD146, NG2, CD34, vWF, and CD271; (c) CD34, CD117, CD146, CD271, NG2, negative for vWF Sox10, Snai1, Slug, Tyrp1, and Oct; which: (a) exhibit telomerase activity no greater than that of human fibroblast; (b) do not exhibit neoplastic growth; (c) are capable of differentiating into three germ layers in vivo and in vitro ; and (d) are capable of self-renewal.
 6. The cell fraction of claim 4 characterized by high phagocytic activity.
 7. The cell fraction of claim 5 characterized by high phagocytic activity.
 8. The cell fraction of claim 4 wherein at least the expression of the CD34 and CD117 cell markers is not observed.
 9. The cell fraction of claim 5 wherein at least the expression of the CD34 and CD117 cell markers is not observed.
 10. The pluripotent cell fraction of claim 8 comprising at least 50% of the SSEA-3 (+) Muse cells.
 11. The pluripotent cell fraction of claim 8 comprising at least 70% of the SSEA-3 (+) Muse cells.
 12. The pluripotent cell fraction of claim 8 comprising at least 90% of the SSEA-3 (+) Muse cells.
 13. The pluripotent cell fraction of claim 8 comprising at least 95% of the Muse cells.
 14. The pluripotent cell fraction of claim 9 comprising at least 50% of the isolated SSEA-3 (+) Muse cells.
 15. The pluripotent cell fraction of claim 9 comprising at least 70% of the isolated SSEA-3 (+) Muse cells.
 16. The pluripotent cell fraction of claim 9 comprising at least 90% of the isolated SSEA-3 (+) Muse cells.
 17. The pluripotent cell fraction of claim 9 comprising at least 95% of the SSEA-3 (+) Muse cells.
 18. The pluripotent cell fraction of claim 13 which is positive for Nanog, Oct3/4, SSEA-3, and Sox2.
 19. The pluripotent cell fraction of claim 17 which is positive for Nanog, Oct3/4, SSEA-3, and Sox2.
 20. The pluripotent cell fraction of claim 13 which is capable of systemic administration to treat damaged tissue or an organ.
 21. The pluripotent cell fraction of claim 17 which is capable of systemic administration to treat damaged tissue or an organ. 